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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes

Department of Medicine, Baylor College of Medicine, Houston, Texas 77030

Submitted 3 April 2003 ; accepted in final form 24 May 2003

	ABSTRACT

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Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H₂O₂ stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C₂C₁₂ myotubes with 100 µM H₂O₂ increased the rate of ¹²⁵I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H₂O₂ and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H₂O₂ stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H₂O₂ upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E2_14k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system.

cachexia; proteolysis; reactive oxygen species; free radicals; aging

In this pathway, ubiquitin conjugation to a protein substrate initiates degradation of the protein by the 26S proteasome complex. The ubiquitin-activating enzyme (E1 protein) activates ubiquitin, which is then transferred to a ubiquitin carrier protein (E2). The E2 protein interacts with a ubiquitin ligase (E3) to catalyze transfer of ubiquitin to a protein substrate, marking the substrate for proteasomal degradation as ubiquitin accumulates. Specificity of the system is attributed to the E2 and E3 proteins that target particular proteins for degradation (13). For example, in skeletal muscle E2_14K and E3 work together to transfer ubiquitin to protein substrates that contain basic and large hydrophobic NH₂-terminal amino acids, termed the "N-end rule pathway" (14). Subsequent research identified additional E2 and E3 proteins that may contribute to muscle atrophy in catabolic conditions. These include the E2 protein UbcH2/E2_20K (16) and the E3 proteins atrogin1/MAFbx (2, 6) and MuRF1 (2). ROS effects on expression of the genes that code for these E2 and E3 proteins have not been established.

The current study addressed ROS effects on ubiquitin conjugation in muscle cells. Specifically, our experiments tested two hypotheses. The first hypothesis was that ROS stimulate ubiquitin-conjugating activity in skeletal muscle cells. The elevated levels of ubiquitin carrier protein previously measured in ROS-exposed muscle (7) could increase the rate at which ubiquitin is conjugated to protein substrates, i.e., ubiquitin-conjugating activity. To test this possibility, we exposed differentiated myotubes from the skeletal muscle-derived C₂C₁₂ cell line to H₂O₂ in cell culture. We subsequently measured the magnitude and time course of changes in integrated activity of the ubiquitin-conjugating pathway. The second hypothesis was that ROS upregulate expression of genes for E2 and E3 proteins that regulate ubiquitin conjugation during muscle wasting. Among the hundreds of E2 and E3 proteins expressed by mammalian cells, several have been identified as putative mediators of skeletal muscle catabolism. These include two E2 proteins (E2_14k, UbcH2) and three E3 proteins (E3, atrogin1/MAFbx, and MuRF1). We used Northern blot analysis to test for increased expression of these genes in C₂C₁₂ myotubes after H₂O₂ stimulation.

	METHODS

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Myogenic cell cultures. Myoblasts from the mouse skeletal muscle cell line C₂C₁₂ (American Type Culture Collection, Rockville, MD) were cultured as described previously (17). In brief, C₂C₁₂ cells were cultured in DMEM supplemented with 10% fetal calf serum and gentamicin at 37°C in the presence of 5% CO₂. Myoblast differentiation was initiated by replacing the growth medium with differentiation medium, DMEM supplemented with 2% heat-inactivated horse serum. Differentiation was allowed to continue for 96 h before experimentation, changing to fresh medium at 48 h. H₂O₂ (Sigma, St. Louis, MO) was incubated with myotubes in DMEM supplemented with 0.5% heat-inactivated horse serum. Cycloheximide or actinomycin D (Sigma) was preincubated with myotubes for 30 min before H₂O₂ was added.

Assay for ubiquitin-conjugating activity. Whole cell extracts of C₂C₁₂ myotubes were made by three freeze-thaw cycles followed by 30-min incubation at 4°C in buffer C containing (in mM) 20 Tris · HCl pH 7.9, 420 NaCl, 1.5 MgCl₂, 0.2 EDTA, 0.5 DTT, and 0.2 PMSF with 25% glycerol, 1 µg/ml leupeptin, and 2 µg/ml aprotinin. The extracts were dialyzed against a buffer containing 10 mM, Tris · HCl, pH 7.6, 1 mM DTT, and 10% glycerol. ¹²⁵I-labeled ubiquitin was prepared by iodination of ubiquitin (Sigma) with carrier-free Na¹²⁵I (Amersham Pharmacia). Dialyzed extracts (100 µg protein) were incubated with ¹²⁵I-ubiquitin (100,000 cpm) in a buffer containing 20 mM Tris · HCl, pH 7.6, 20 mM KCl, 5 mM MgCl₂, 1 mM DTT, 10% glycerol, 30 µM MG-132 (to inhibit proteasome activity), 1 µM ubiquitin aldehyde (to inhibit the hydrolysis of ubiquitin conjugates by deubiquitinating isopeptidases), and 2 mM 5'-adenylylimidodiphosphate (AMPPNP). AMPPNP is an ATP analog that provides energy for activation of ubiquitin by E1 but not for degradation of ubiquitinated proteins by the proteasome (10). The reaction mixtures (20-µl total volume) were incubated at 37° C for 60 min, after which the reaction was terminated by addition of 20 µl of 2x Laemmli sample buffer (12). The mixture was heated to 90°C for 3 min and separated on SDS-PAGE (15% gel). To quantify ubiquitination activity, autoradiographs of the gel were analyzed by densitometry software (ImageQuant 5.2, Molecular Dynamics).

Northern blot analysis. Total RNA was isolated from C₂C₁₂ myotubes by use of the RNAzol reagent (TEL TEST). Twelve micrograms of each sample was separated by agarose gel electrophoresis modified from the procedure described by Liu and Chou (18). Briefly, RNA samples were denatured by heating to 65°C for 3 min in a sample loading buffer containing 1x Tris-boric acid-EDTA (TBE), 6.5% Ficoll, 0.005% bromphenol blue, 0.025% xylkene cyanol ff, and 3.5 M urea (final concentrations). The samples were immediately chilled on ice and separated with 1% native agarose gel at 8 V/cm. RNA was then blotted and ultraviolet cross-linked to a Gene-Screen membrane (NEN Life Science Products, Boston, MA). Prehybridization (4 h) and hybridization (16 h) were carried out in ULTRAhyb buffer (Ambion) at 42°C. Hybridization probes were full-length human coding sequences of the specific genes except that the polyubiquitin probe was of mouse origin. The probes were prepared from isolated total RNA with a one-step RT-PCR kit (Promega, Madison, WI) and were labeled with [-³²P]dCTP (3,000 Ci/mmol, Amersham Pharmacia, Arlington Heights, IL) by using the random primer method. After hybridization, each membrane was washed and exposed to X-ray film. Levels of mRNA were quantified by analyzing autoradiographs with densitometry software (ImageQuant).

Electrophoresis mobility shift assay. Nuclear extracts of C₂C₁₂ myotubes were prepared as described previously (17) with buffer C modified by the addition (in mM) of 30 sodium pyrophosphate, 5 Na₃VO₄, 2 iodoacetic acid, 50 NaCl, 50 NaF, and 5 ZnCl₂. Electrophoresis mobility shift assay (EMSA) was carried out as previously described (17). Briefly, the binding assay buffer contained (in mM) 5 Tris · HCl, pH 7.5, 100 NaCl, 0.3 DTT, and 5 MgCl₂ with 10% glycerol, 2 µg of bovine serum albumin, 0.2% NP-40, and 1 µg of polydeoxyinosinic-deoxycytidylic acid [poly(dI-dC) · poly(dI-dC)]. A DNA probe containing the NF-B consensus binding site (5'-AGTTGAGGGGACTTTCCCAGGC-3'; consensus binding site underlined) was labeled with [-³²P]dATP (3,000 Ci/mmol, Amersham Pharmacia, Arlington Heights, IL) with the Klenow fragment. After 20-min preincubation of 10 µg of nuclear extract, 1 ng (10,000-15,000 cpm) of labeled probe was added and incubation was continued for 30 min on ice; the reaction mixtures were resolved on 4.5% polyacrylamide gels. Protein concentration of the nuclear extracts was determined by the Bradford method (Bio-Rad protein assay kit).

Statistical analysis. Densitometry data sets were tested for normality and equal variance with commercial software (SigmaStat, SPSS Science, Chicago, IL). Data were then evaluated with one-way analysis of variance combined with Tukey's multiple-comparison test (29). Differences between groups were considered significant at the P < 0.05 level. Values are reported as means ± SE.

	RESULTS

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H₂O₂ and ubiquitin-conjugating activity. As shown in Fig. 1, myotubes possess a basal level of ATP-dependent ubiquitin-conjugating activity that is detectable before H₂O₂ stimulation. This activity remains unaltered 1 h after exposure to 100 µM H₂O₂ but becomes elevated within 4 h (Fig. 1) and remains elevated for at least 24 h (data not shown). Control studies confirmed that formation of ubiquitin conjugates requires inclusion of the ATP analog AMPPNP (Fig. 1) and the isopeptidase inhibitor ubiquitin aldehyde (isopeptidase removes conjugated ubiquitin from proteins; data not shown). The ability of H₂O₂ to stimulate ubiquitin-conjugating activity was abolished by preincubating myotubes with 20 µg/ml cycloheximide, a protein synthesis inhibitor, or 1 µg/ml actinomycin D, an RNA synthesis inhibitor (Fig. 2). Activity also appeared to be increased by cycloheximide alone. The direct effect of H₂O₂ on ubiquitin-conjugating activity was assessed with a cell-free system. Whole cell extracts were incubated with 40, 100, or 250 µMH₂O₂ for 1 h and then were tested for ubiquitin-conjugating activity; H₂O₂ preconditioning had no effect (data not shown). Together, these findings suggested that H₂O₂ effects on ubiquitin-conjugating activity are likely to be regulated at the transcriptional level.

View larger version (27K): [in this window] [in a new window]   Fig. 1. H2O2 stimulates ubiquitin-conjugating activity. C2C12 myotubes were incubated with 100 µM H2O2 for indicated period. Whole cell extracts prepared from harvested myotubes were incubated with 125I-labeled ubiquitin (125I-Ub). A: ubiquitin conjugation to muscle proteins as visualized by autoradiography of SDS-PAGE used to separate the proteins; film depicts composite image obtained from a single film generated in 1 of 3 experiments; conjugation after H2O2 exposure [optical density (OD) = 172% control at both 4 and 6 h] illustrates responses seen in each of 3 experiments. AMPPNP, 5'-adenylylimido-diphosphate. B: average data as quantified by densitometry. The overall P value shown was obtained by ANOVA; *difference from control (P < 0.05) determined by Tukey's multiple-comparison test.

View larger version (95K): [in this window] [in a new window]   Fig. 2. Cycloheximide and actinomycin D block H2O2 effects on ubiquitin-conjugating activity. C2C12 myotubes were preincubated with 20 µg/ml of cycloheximide or 1 µg/ml of actinomycin D for 30 min before incubation with or without 100 µM H2O2 for 6 h. Autoradiograph depicts a composite image obtained from a single film illustrating 125I-ubiquitin conjugation into muscle proteins.

H₂O₂ and E2/E3 gene expression. We then evaluated H₂O₂ effects on the expression of genes that regulate ubiquitin-conjugating activity during muscle wasting. Changes in mRNA levels were determined by Northern blot analysis. Because the polyubiquitin gene is generally upregulated by stimuli that increase ubiquitin-conjugating activity, we first examined H₂O₂ effects on polyubiquitin expression. As shown in Fig. 3, C₂C₁₂ myotubes incubated with 100 µM H₂O₂ exhibited a 19% increase in polyubiquitin mRNA after 6 h. We subsequently evaluated mRNA levels for E2 and E3 proteins that are reported to regulate muscle catabolism. Each E2 and E3 had a detectable level of constitutive expression, but only a subset responded to H₂O₂ stimulation. We observed a 29% increase in E2_14k mRNA levels 6 h after H₂O₂ stimulation (Fig. 4), which is consistent with the findings of Gomes-Marcondes and Tisdale (7). In contrast, there was no change in UbcH2/E2_20k mRNA levels during this period (data not shown) and H₂O₂ did not alter E3 expression (data not shown). However, other E3 genes responded to H₂O₂ stimulation. Atrogin1/MAFbx expression was increased 42% after 3-h incubation (Fig. 5), and MuRF1 expression was increased 46% after 6 h (Fig. 6). These observations confirm that H₂O₂ upregulates genes that regulate ubiquitin conjugation and suggest that this transcriptional response involves a subset of ROS-responsive genes.

View larger version (45K): [in this window] [in a new window]   Fig. 3. H2O2 upregulates the polyubiquitin gene. C2C12 myotubes were incubated with 100 µM H2O2 for indicated period. Total RNA was isolated for Northern blot analysis with full-length cDNA probes. A: autoradiograph from 1 of 3 experiments; polyubiquitin mRNA increased 6 h after H2O2 exposure (OD = 130% time 0). B: photograph of corresponding ethidium bromide-stained agarose gel (loading control). C: averaged data quantified by densitometry; means ± SE are shown. ANOVA indicated an overall difference among groups (P < 0.05); *difference from time 0 as analyzed by Tukey's multiple-comparison test (P < 0.05).

View larger version (47K): [in this window] [in a new window]   Fig. 4. H2O2 upregulates E214k. C2C12 myotubes were incubated with 100 µM H2O2 for indicated period. Total RNA was isolated for Northern blot analysis with full-length cDNA probes. A: autoradiograph from 1 of 3 experiments depicts increase in E214k mRNA 6 h after H2O2 exposure (OD = 139% time 0). B: photograph of corresponding ethidium bromide-stained agarose gel (loading control). C: averaged data quantified by densitometry; means ± SE are shown. ANOVA indicated a significant difference among groups; *difference from control as analyzed by Tukey's multiple-comparison test (P < 0.05).

View larger version (43K): [in this window] [in a new window]   Fig. 5. H2O2 upregulates atrogin-1/MAFbx. C2C12 myotubes were incubated with 100 µM H2O2 for indicated period. Total RNA was isolated for Northern blot analysis with full-length cDNA probes. A: autoradiograph from 1 of 5 experiments depicts increase in atrogin-1/MAFbx mRNA at 3 h (OD = 170% time 0) and 6 h (165%). B: photograph of corresponding ethidium bromide-stained agarose gel (loading control). C: averaged data quantified by densitometry; means ± SE are shown. ANOVA indicated an overall difference among groups; *difference from time 0 by Tukey's multiple-comparison test (P < 0.05).

View larger version (39K): [in this window] [in a new window]   Fig. 6. H2O2 upregulates MuRF1. C2C12 myotubes were incubated with 100 µM H2O2 for indicated period. Total RNA was isolated for Northern blot analysis with full-length cDNA probes. A: autoradiograph shows increase in MuRF1 mRNA seen at 6 h (168% time 0) in 1 of 3 experiments. B: photograph of corresponding ethidium bromide-stained agarose gel (loading control). C: averaged data quantified by densitometry; means ± SE are shown. Overall difference among groups was detected by ANOVA; *difference from time 0 by Tukey's multiple-comparison test (P < 0.05).

We were puzzled by the failure of UbcH2 to respond to H₂O₂ because TNF- appears to upregulate the UbcH2 gene via ROS-dependent NF-B activation (15, 16). We therefore conducted follow-up studies to test the effect of H₂O₂ exposure on NF-B activity. As illustrated in Fig. 7, incubation with H₂O₂ increased NF-B translocation to myotube nuclei, which confirmed prior reports (16). However, this response was dose dependent and relatively weak. H₂O₂ concentrations >100 µM were required to evoke detectable NF-B activation, and the magnitude of this response was less than the NF-B activation stimulated by 6 ng/ml TNF-. These data suggest that in prior experiments (above) 100 µM H₂O₂ did not activate NF-B and therefore did not upregulate UbcH2 expression.

View larger version (52K): [in this window] [in a new window]   Fig. 7. H2O2 is a weak activator of NF-B. C2C12 myotubes were incubated with TNF- for 30 min or with various concentrations of H2O2 for 2 h. These incubation times were previously demonstrated to yield maximal activation of NF-B by the 2 reagents (17). Nuclear extracts of the myotubes were analyzed by electrophoresis mobility shift assay (EMSA) with a NF-B probe.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
This study contributes to our understanding of the cellular and molecular mechanisms by which ROS might promote skeletal muscle catabolism. The current data provide the first direct evidence that H₂O₂ increases ubiquitin-conjugating activity in skeletal muscle cells. The data further demonstrate that H₂O₂ exposure can alter the expression of genes that regulate this pathway, including the polyubiquitin gene and genes for key E2 and E3 proteins. The biological significance of these observations is briefly outlined below.

ROS and muscle catabolism. Like all eukaryotic cells, mammalian skeletal muscle cells continuously generate ROS, which are detectable in the cytosol (23) and are released into the extracellular space (25). The physiological level of H₂O₂ in mammalian tissue is believed to be in the range of several micromoles per liter (4). Increased ROS activity and oxidative stress are closely associated with loss of muscle mass in catabolic states that include cancer (28), chronic obstructive pulmonary disease (9), congestive heart failure (1), aging-induced sarcopenia (27), and immobilization (11). To model this process in a cell culture system, we selected a concentration of H₂O₂ (100 µM) that is not toxic to muscle cells (25) and stimulates loss of muscle protein (7). Gomes-Marcondes and Tisdale (7) showed that C₂C₁₂ myotubes exposed to 100 µM H₂O₂ exhibit an increase in total protein catabolism as measured by [³H]phenylalanine release. They also detected increases in proteasomal activity, 20S proteasome content, and E2_14k protein levels, leading them to conclude that H₂O₂ induces catabolism via the ubiquitin-proteasome pathway.

The role of ubiquitin conjugation. Protein degradation by the ubiquitin-proteasome pathway is influenced by the rate of ubiquitin conjugation to targeted substrates and the catalytic activity of 26S proteasome complexes (22). Our current study focused on the regulation of ubiquitin conjugation, a complex, ATP-dependent process that requires interaction among E1, E2, and E3 proteins. We used the method of Lecker and colleagues (14) to measure the integrated activity of this multienzyme pathway in whole cell extracts. Ubiquitin-conjugating activity was increased in myotubes that were incubated with H₂O₂ in cell culture but not in whole cell extracts directly exposed to H₂O₂. These findings argued against a posttranslational mechanism of action for H₂O₂.

A transcriptional mechanism seemed more likely. Gomes-Marcondes and Tisdale (7) had already shown that E2_14k protein levels are elevated 24 h after H₂O₂ exposure. Their finding suggested that H₂O₂ might stimulate ubiquitin-conjugating activity by increasing the expression of one or more regulatory enzymes in the pathway. Consistent with this model, we found a 4-h lag between H₂O₂ exposure and the subsequent increase in ubiquitin-conjugating activity. Moreover, this increase could be blocked by cycloheximide or actinomycin D. These findings suggested that H₂O₂ stimulates ubiquitin conjugation by altering muscle gene expression.

Transcriptional regulation of the process. Subsequent experiments determined that H₂O₂ upregulates genes that are known to affect ubiquitin conjugation during muscle atrophy. These include the polyubiquitin gene. Ubiquitin availability is not generally considered a rate-limiting factor in ubiquitin conjugation. However, expression often is upregulated when ubiquitin-conjugating activity is elevated (5, 19-21). This may reflect a compensatory response that maintains adequate ubiquitin supplies under conditions of increased utilization.

Among the regulatory enzymes upregulated by H₂O₂, E2_14k is a critical regulator of ubiquitin conjugation via the N-end rule (14). E2_14k is thought to interact with E3 to mediate muscle wasting in various catabolic states (13). The E3 proteins atrogin1/MAFbx and MuRF1 also appear to play important roles in muscle atrophy. Recent evidence implicates both of these E3 proteins in experimental models of catabolism that include fasting, diabetes, cancer, renal failure, hindlimb suspension, immobilization, and denervation (2, 6).

The timing of these responses is of interest. The mRNA for atrogin1/MAFbx was elevated at 3 h, much earlier than the 6-h response time exhibited by other H₂O₂-responsive genes, suggesting a distinct mechanism of regulation for atrogin1/MAFbx expression. These transcriptional events also appear to have persistent effects on cell function. Twenty-four hours after H₂O₂ exposure, Gomes-Marcondes and Tisdale (7) measured elevated E2_14K levels and we observed continuing elevation of ubiquitin-conjugating activity.

Subsequent experiments tested H₂O₂ effects on NF-B, a redox-sensitive transcription factor that regulates UbcH2/E2_20K expression (16). We found no evidence that 100 µM H₂O₂ stimulates NF-B activation under the current experimental conditions. This explains the failure of UbcH2/E2_20K mRNA to increase in myotubes after H₂O₂ exposure. Furthermore, these results suggest that NF-B is not an essential regulator of the genes that did respond to H₂O₂, including polyubiquitin, E2_14k, atrogin1/MAFbx, and MuRF1.

In conclusion, H₂O₂ exposure appears to promote protein degradation in skeletal muscle via the ubiquitin-proteasome pathway. Results of the current study suggest that muscle catabolism is due, at least in part, to ROS effects on the multienzyme process by which ubiquitin is conjugated to protein substrates. Oxidative signaling appears to activate a subset of redox-sensitive genes that code for ubiquitin and regulatory E2 and E3 proteins. Upregulation of these gene products increases the rate of ubiquitin conjugation within skeletal muscle, a persistent response that accelerates the targeting of muscle proteins for degradation by the 26S proteasome.

	ACKNOWLEDGMENTS

 
This research was supported by National Heart, Lung, and Blood Institute Grant HL-59878, the National Space Biomedical Research Institute, and the Muscular Dystrophy Association.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. B. Reid, Dept. of Medicine, Baylor College of Medicine, One Baylor Plaza, Suite 520B, Houston, TX 77030 (E-mail: reid{at}bcm.tmc.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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