Department of Medicine, Baylor College of Medicine, Houston, Texas 77030
Submitted 3 April 2003 ; accepted in final form 24 May 2003
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ABSTRACT |
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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 E214K 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/E220K
(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 (E214k, 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.
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METHODS |
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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.
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RESULTS |
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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
E214k 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/E220k 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.
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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.
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DISCUSSION |
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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 E214k 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 E214k 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,
E214k is a critical regulator of ubiquitin conjugation via the
N-end rule (14).
E214k 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 E214K 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/E220K 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/E220K 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, E214k,
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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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