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increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k
Department of Medicine, Baylor College of Medicine, Houston, Texas, USA;
* Renal Unit, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA;
AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, England, SK10 4TG; and
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA
1Correspondence: Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Suite 520B, Houston, TX 77030, USA. E-mail: reid{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is thought to stimulate muscle catabolism via an NF-
B-dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF- stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF-B binding site; NF-B binding to this sequence is increased by TNF- stimulation. A dominant negative inhibitor of NF-B activation blocks both UbcH2 up-regulation and the increase in ubiquitin-conjugating activity stimulated by TNF-. In extracts from TNF--treated myotubes, ubiquitin-conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild-type UbcH2. Thus, UbcH2 up-regulation is a novel response to TNF-/NF-B signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.—Li, Y.-P., Lecker, S. H., Chen, Y., Waddell, I. D., Goldberg, A. L., Reid, M. B. TNF- increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k.
Key Words: cachexia • catabolism • atrophy • cytokines • nuclear factor B
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) is a proinflammatory cytokine thought to stimulate muscle atrophy in various diseases (1
2
3
4
5)
and in aging (6)
. Studies of cultured myotubes indicate that the transcription factor nuclear factor B (NF-B) plays a pivotal role in the catabolic response to TNF- (7)
. In skeletal muscle, TNF- activates NF-B via a rapid, dose-dependent process that requires degradation of the inhibitory protein I-B (8
9
10
11
12
13)
. Once I-B is degraded, NF-B is translocated to the myocyte nucleus where it alters gene expression, stimulating protein loss in differentiated muscle cells (8
, 14)
.
TNF- appears to stimulate general proteolysis by increasing ubiquitin conjugation to muscle proteins (7)
. In this process, the ubiquitin-activating enzyme (E1 protein) activates ubiquitin, which is then transferred to a ubiquitin carrier protein (E2). The ubiquitin carrier protein interacts with a ubiquitin ligase (E3) to catalyze transfer of ubiquitin to the protein substrate, marking the substrate for proteasomal degradation as ubiquitin accumulates (15)
. Several lines of evidence indicate that TNF- stimulates this process. Diseases that cause an increase in serum TNF- levels are associated with increased ubiquitin mRNA in skeletal muscle (16)
. Intravenous administration of TNF- to rodents increases ubiquitin content and ubiquitin-conjugated protein levels in muscle (17)
, and in vitro incubation with TNF- increases ubiquitin mRNA in rat limb muscle (18)
. Despite this association, a transcriptional mechanism by which TNF- might stimulate ubiquitin conjugation has yet to be identified.
We therefore screened differentiated C2C12 myotubes for changes in gene expression in response to TNF- exposure. We detected a rapid rise in the mRNA for UbcH2 (NCBI accession #P37286), an E2 protein first identified in human placenta (19)
and later described as E220k in murine tissue (20)
. The mouse homologue is constitutively expressed in skeletal muscle (21)
, but the expression pattern in human tissue and the function of this protein in muscle are not known. The current project addressed the function of UbcH2 in TNF--exposed muscle by testing two hypotheses: 1) UbcH2 gene expression in skeletal muscle is stimulated by TNF-/NF-B signaling, and 2) UbcH2 up-regulation is important for the general increase in ubiquitin-conjugating activity that occurs in TNF--exposed muscle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. C2C12 cells were cultured in DMEM supplemented with 20% newborn 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). Limb skeletal muscles were excised from neonatal rats (2–4 days old), minced with razor blades in a minimal volume of PBS, and enzymatically dissociated in dissociation buffer (0.1% trypsin, 0.1% collagenase type 2, and 0.025% DNase in PBS) at 37°C for 5 min. The slurry was centrifuged in 20 mL DF20 (20% fetal bovine serum in DMEM) at 150 x g for 1 min at 4°C and the supernatant was discarded. The dissociation process was repeated three times. Each time the pelleted cells were incubated in dissociation buffer for 15 min and the supernatant was collected by centrifuging at 300 x g for 7 min at 4°C. The dissociated cells were pelleted and resuspended in 1.082 g/mL Percoll (Pharmacia, Piscataway, NJ, USA) for purification through a density gradient (1.050, 1.060, and 1.082 g/mL) by centrifugation at 2000 x g for 25 min at room temperature. The Percoll gradient was made in a buffer containing 6.8 g/L NaCl, 0.4 g/L KCl, 0.1 g/L MgSO4, 1.5 g/L NaH2PO4, 1.0 g/L dextrose, and 4.76 g/L HEPES (pH 7.3). The band containing myocytes at the interface between 1.060 and 1.082 g/mL Percoll layers was collected and washed twice in the gradient buffer plus 0.02 g/L Phenol Red. The cells were resuspended in Ham's F-10 nutrient mixture (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 20% fetal calf serum, then plated in Primaria dishes (Becton Dickinson and Co., Franklin Lakes, NJ, USA) and grown at 37°C in the presence of 5% CO2. Differentiation was induced as described above for C2C12 cells. The culture media was changed at 24 h intervals. Cells were exposed to TNF- at the time of media change by adding mouse recombinant TNF- (Roche, Indianapolis, IN, USA) to achieve final concentrations of 3 or 6 ng/mL. These levels fall within the range of circulating TNF- levels measured in patients (22
, 23)
and induce loss of muscle protein in mature myotubes (8
, 14)
. In some experiments, myotubes were treated with 100 µM pyrrolidine dithiocarbamate (PDTC; Sigma Aldrich, St. Louis, MO, USA) 30 min before TNF- exposure and throughout the exposure period.
Animal use
Experimental protocols were approved in advance by the Animal Protocol Review Committee of the Baylor Animal Program. Adult ICR mice were preconditioned by intraperitoneal (i.p.) injection of TNF- 100 µg/kg body weight or an equal volume of diluent. Animals were deeply anesthetized 2–4 h later by i.p. injection of pentobarbital sodium 85 mg/kg. Gastrocnemius muscles were excised for study and the anesthetized animals were killed by rapid exsanguination.
Northern blot analyses
Total RNA was isolated from rat or C2C12 myotubes by using the RNAzol reagent (TEL TEST). Twelve micrograms of each sample were separated with agarose gel electrophoresis modified from the procedure described by Liu and Chou (24)
. RNA samples were denatured by heating to 65°C for 3 min in a sample loading buffer containing 1x 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 with a running time of slightly less than 3 h at 8V/cm. RNA was then blotted and UV cross-linked to GeneScreen membrane (NEN Life Science Products, Boston, MA, USA). Prehybridization (4 h) and hybridization (16 h) were performed at 45°C in a buffer containing 50% deionized formamide, 5x SSPE, 5x Denhardt's, 1% SDS, 200 µg/mL salmon sperm DNA, and 10% dextran sulfate (Pharmacia). Hybridization probes were full-length cDNAs of the specific genes that were generated by RT-PCR and labeled with [-32P]dCTP (3000 Ci/mmol, Amersham Pharmacia, Arlington Heights, IL, USA) using the random primer method. A membrane blotted with RNA isolated from various human tissues was purchased from Clontech (Palo Alto, CA, USA). After hybridization, the membranes were washed and exposed to X-ray films. Levels of mRNA were quantified by analyzing autoradiographs using densitometry software (ChemiDoc, Bio-Rad, Hercules, CA, USA).
Western blot analyses
Western blots were performed as described previously (8)
. Cell lysates were prepared by boiling harvested cells in Laemmli buffer (25)
for 5 min, separated with SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were incubated in the presence of rabbit antiserum raised against recombinant UbcH2. Horseradish peroxidase-conjugated secondary antibodies were used to locate the primary antibodies. Antibodies were visualized by the enhanced chemiluminescence method (Amersham). The detected bands on the X-ray films were quantified using densitometry software (ChemiDoc, Bio-Rad). Protein concentration in the cell lysates was determined using a Bio-Rad Dc protein assay kit.
Recombinant proteins and antiserum
For synthesis of dominant negative UbcH2 (C87S-UbcH2), cys-87 of the UbcH2 cDNA was mutated to serine by PCR using the overlapping extension method (26)
. For overexpression of wild-type and mutant UbcH2 proteins, the corresponding cDNAs were subcloned into the expression vector pProEX-HTb (Invitrogen Life Technology, San Diego, CA, USA), amplified in the DH-5 strain of Escherichia coli, and purified from the bacteria lysates using a 6xHis column (Pierce Chemical Co., Rockford, IL, USA). Antiserum was generated by a commercial firm (Sigma-Genosys, Woodlands, TX, USA) that we provided with purified, recombinant wild-type UbcH2 protein. Preimmune serum was obtained from two rabbits. The animals then were injected with UbcH2 on days 0, 14, 28, 42, 56, and 70. Antiserum was collected on days 49, 63, and 77. Immunoglobulin G was isolated from antiserum or preimmune serum on a protein A column by use of a commercial purification kit (Pierce).
Electrophoresis mobility shift assay (EMSA)
EMSA was performed as described previously (8)
. The binding assay buffer contained 5 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.3 mM dithiothreitol, 5 mM MgCl2, 10% glycerol, 2 µg bovine serum albumin, 0.2% NP-40, and 1 µg of poly (dI-dC). A 20-mer DNA probe made up of nucleotides -749 to -730 of a 5' flanking sequence of the UbcH2 gene containing a consensus NF-B binding site (5'-GGTGTGGGGATTTCCAGATT-3') was labeled with [-32P]dATP (3000 Ci/mmol, Amersham Pharmacia) using the Klenow fragment. Nuclear extracts were prepared according to Andrews and Faller (27)
. We preincubated 5 µg of nuclear extract in reaction buffer for 20 min; 1 ng (10,000 to 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 concentrations of the nuclear extracts were determined by a commercial assay kit (Bio-Rad). Supershift assays were performed using commercial antibodies for p65 and p50 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
In vitro assay of ubiquitin-conjugating activity
Whole-cell extracts of myotubes (C2C12 or rat primary) were made by 3 freeze-thaw cycles, followed by 30 min incubation at 4°C in a buffer containing 20 mM Tris-HCl pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 1 µg/mL leupeptin, and 2 µg/mL aprotinin. The crude extract was either assayed directly or fractionated using DEAE-cellulose (Whatman DE-32) chromatography; fraction II was eluted from the resin-bound material using 0.5 M NaCl (28)
. Either the whole-cell extract or fraction II was dialyzed against a buffer containing Tris-HCl, pH 7.6, 1 mM DTT, and 10% glycerol. The dialyzed sample (50–100 µg protein) was incubated with 125I-ubiquitin (
100,000 cpm, Amersham Pharmacia) in a buffer containing 20 mM Tri-HCl, pH 7.6, 20 mM KCl, 5 mM MgCl2, 2 mM AMPPNP (an ATP analog that supports protein ubiquitination but not proteasome activity), 1 mM DTT, 10% glycerol, 30 µM MG-132 (proteasome inhibitor), and 1 µM ubiquitin aldehyde (an isopeptidase inhibitor that inhibits disassembly of ubiquitin conjugates). In some experiments, ubiquitin conjugation to specific proteins was assessed by combining purified E1, UbcH2 or E214k, and the protein of interest; we used the incubation system described above minus the cell extract and ubiquitin aldehyde (28)
. 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. The mixture was heated to 90°C for 3 min and separated on SDS-PAGE (15% gel). To quantify ubiquitin-conjugating activity, autoradiographs of the gel were analyzed by densitometry.
Statistics
To assess differences between two conditions, densitometry data sets were tested for normality and equal variance using commercial software (SigmaStat, SPSS Science, Chicago, IL, USA). Data were then evaluated using Student's paired t test for individual comparisons or one-way analyses of variance to assess dose and time dependence (29)
. Differences between groups were considered significant at the P < 0.05 level. Values are reported as mean ± SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, mRNA levels were highest in skeletal and cardiac muscles. Significant UbcH2 expression was also detected in kidney and placenta; liver, brain, lung, and spleen contained lower levels. At least six transcripts of the UbcH2 gene were evident. These were 5.5, 3, 2.8, 2, 1.4, and 1.2 kb in size, each theoretically large enough to transcribe the 183 amino acids that compose the UbcH2 protein. The 5.5 kb transcript predominated, but relative abundance of the transcripts varied among tissues.
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The response of UbcH2 to TNF- was initially tested using primary myocytes isolated from rat limb muscle (Fig. 2
A). Northern blot analysis detected constitutive transcripts of 5.5, 3.0, and 1.4 kb. The two larger transcripts were present at levels too low to quantify reliably but changes in expression of the 1.4 kb transcript could be measured by densitometry. Incubation with TNF- 6 ng/mL for 1.5 h increased cellular content of the 1.4 kb transcript 1.47 ± 0.1-fold (n=3, P<0.05), a transient response that spontaneously reversed by 3 h. This response was not common to all E2 proteins; TNF- did not increase mRNA levels for E214k, CDC34, or UbcH7 (data not shown). As shown in Fig. 2B
, similar increases in UbcH2 mRNA content were seen in the muscles of adult mice treated with TNF- 100 ng/g. UbcH2 mRNA levels increased progressively in gastrocnemius over the 4 h after i.p. injection of the cytokine. We subsequently examined changes in UbcH2 protein levels using differentiated C2C12 myotubes incubated with TNF- 6 ng/mL. As shown in Fig. 2C
, UbcH2 protein with apparent molecular weight of 21 kb was detected. UbcH2 protein levels were not altered 1 h after TNF- exposure but increased 3.6 ± 0.12-fold at 4 h (n=3; P<0.05). The time course of this response is consistent with transcriptional regulation of UbcH2 expression.
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NF-B mediates UbcH2 up-regulation by TNF-
We tested whether NF-B mediates the up-regulation of UbcH2 by use of I-B
N, a dominant negative protein that inhibits NF-B activation in response to TNF- (14)
. As shown in Fig. 3
A, TNF- stimulated UbcH2 expression in control myotubes transfected with the empty viral vector. However, TNF- did not alter UbcH2 expression in dominant negative myotubes that overexpress I-BN, indicating NF-B regulates the UbcH2 gene.
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We subsequently evaluated NF-B sensitivity of the UbcH2 promoter region. Computer software (URL www.cbil.upenn.edu/cgi-bin/tess) was used to identify a consensus NF-B binding site, GGGGATTTCC, in the human gene located 700 nucleotides upstream of the 5' flanking sequence. We tested the capacity of this DNA motif to participate in TNF-/NF-B signaling by EMSA. An oligonucleotide replicating the putative NF-B binding site was used to probe nuclear extracts from untreated C2C12 myotubes and from C2C12 myotubes incubated with TNF- 3 ng/mL for 30 min. As illustrated in Fig. 3B
, extracts from TNF--treated myotubes formed larger amounts of oligonucleotide–protein complex than extracts from control myotubes. Supershift assays confirmed that both p50 and p65 subunits of the NF-B heterodimer were present in the complex.
TNF-/NF-B signaling stimulates ubiquitin-conjugating activity
The method of Lecker and colleagues (30)
was used to measure ubiquitin-conjugating activity in whole-cell extracts prepared from C2C12 and rat primary myotubes. As shown in Fig. 4
, differentiated myotubes exhibit a low level of constitutive ubiquitin-conjugating activity that is increased by TNF- exposure. The response to TNF- is dose-dependent (n=3; P<0.01), as illustrated in Fig. 4A
. Data in Fig. 4B
depict the time course of this response. Activity is unaltered by a 1 h exposure to TNF- but is increased after 4–6 h (n=3; P<0.001), remaining elevated for at least 24 h. Increased activity was also observed in rat primary myotubes exposed to TNF- 6 ng/mL (data not shown). Control studies confirmed that formation of ubiquitin conjugates requires inclusion of an ATP analog (Fig. 4A
) and an isopeptidase inhibitor (data not shown) in the assay buffer. Fraction II extracts from C2C12 myotubes, i.e., the ubiquitin-free fraction of cell proteins that bind DEAE cellulose (28)
, exhibited a similar response to TNF- (Fig. 4C
) without the low molecular weight bands seen in whole-cell extracts. Thus, the key E2 and E3 enzymes altered by TNF- appear to be present in fraction II.
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We next tested whether NF-B mediates TNF- effects on ubiquitin-conjugating activity. Results obtained using a dominant negative approach are illustrated in Fig. 5
A. TNF- increased ubiquitin-conjugating activity in control myotubes containing the empty pCMV vector but did not alter activity in myotubes that overexpressed I-BN. In a second series of experiments, C2C12 myotubes were treated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-B (31)
. Figure 5B
illustrates the effect of PDTC which partially prevented the rise in ubiquitin-conjugating activity stimulated by TNF- (n=3; P<0.01).
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UbcH2 effects on ubiquitin-conjugating activity
The functional importance of UbcH2 was first tested by immunodepletion studies. Rabbit antiserum was raised against recombinant human UbcH2 and purified. Addition of the antiserum to extracts from TNF--treated myotubes inhibited ubiquitin-conjugating activity by 70% (n=3; P<0.02). Dose-dependent inhibition by anti-UbcH2 is illustrated in Fig. 6
A. Preimmune serum from the same rabbit had no effect.
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We next used a standard dominant negative approach (32)
to test the role of UbcH2 in ubiquitin conjugation. The dominant negative UbcH2 (C87S-UbcH2) was constructed by mutating cys-87 to serine and overexpressing the protein in E. coli. As illustrated in Fig. 6B
, ubiquitin-conjugating activity was inhibited by addition of C87S-UbcH2 to extracts from TNF--treated myotubes (66.3%±6.6 SE; P<0.05). Addition of wild-type UbcH2 had the opposite effect, increasing activity (119 ± 1; P < 0.05). Similar results were obtained using E214k, an E2 protein that mediates ubiquitin conjugation in muscle atrophy (33)
. The dominant negative mutant of E214k decreased activity whereas the wild-type protein increased activity (data not shown). In contrast, activity was unaffected by a dominant negative mutant of E2-C/UbcH10, an E2 that regulates the cell cycle (34)
. This indicates selectivity of E2 action.
The capacity of UbcH2 to transfer ubiquitin directly to protein substrates was tested using an E3-free biochemical system. In the presence of E1 and the ATP analog AMPPNP, UbcH2 catalyzed the conjugation of ubiquitin to histone H2A as reported previously (19
, 35)
. E214k exhibited similar activity. However, neither UbcH2 nor E214k catalyzed detectable conjugation of ubiquitin to myofilament proteins (myosin, actin, troponin, tropomyosin) or to lactalbumin, an N-end rule substrate (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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who noted its sequence homology to yeast UBC8. The amino acid sequence of UbcH2 is identical to its murine homologue E220k (NCBI accession #P37286). The UbcH2 gene is located on chromosome 7 and exhibits a complex expression pattern comprising at least five different mRNA transcripts ranging in size from 0.8 to > 5 kb (19)
. Biochemical studies by Kaiser et al. (19)
demonstrated the capacity of recombinant UbcH2 to conjugate ubiquitin to histone H2A via an E3-independent reaction that required only E1 protein, ATP, and ubiquitin. A follow-up study by this same group (35)
used deletion mutants to evaluate the functional domains of UbcH2. Aside from these reports, little is known about the expression, regulation, or functional importance of this protein in humans.
In mice, constitutive expression of the UbcH2 homologue E220k varies among tissues. mRNA levels are detectable in skeletal and cardiac muscles but are higher in spleen, lung, and brain (21)
. This differs from the pattern of UbcH2 expression in human tissues where skeletal and cardiac muscles appear to have the highest mRNA content. Constitutive expression suggests that UbcH2 plays a role in normal turnover of muscle proteins.
TNF- exposure increased UbcH2 expression both in cultured myotubes and in the muscles of mice injected with the cytokine. Interpretation of the animal studies is complicated by the systemic TNF- effects on hormonal status (36)
and food consumption (37)
, which may alter muscle gene expression indirectly. In contrast, cell culture studies indicate that TNF- acts directly on differentiated myotubes to stimulate UbcH2 expression. UbcH2 mRNA levels were increased after 90 min of TNF- exposure, a response mediated by the transcription factor NF-B. This was not a generic transcriptional response common to all E2 proteins since mRNAs for three other E2 proteins were unaffected by TNF-. We also observed a rise in UbcH2 protein content that trailed the rise in mRNA by 2–3 h. To our knowledge, this is the first demonstration of increased E2 protein levels in muscle after a catabolic stimulus.
TNF- is widely believed to stimulate muscle catabolism by general activation of the ubiquitin/proteasome pathway. Animal studies identify TNF- as a circulating factor that promotes ubiquitin conjugation to muscle proteins in vivo (17
, 38
39
40
41)
. However, as noted above, TNF- has multiple systemic effects that may stimulate muscle catabolism indirectly. The only evidence for a direct effect is the study of Llovera et al. (18)
in which ubiquitin mRNA was elevated in rat soleus muscles after in vitro incubation with TNF-.
The current data demonstrate that TNF- can act directly on differentiated myotubes to stimulate persistent up-regulation of ubiquitin-conjugating activity. This response appears to be regulated at the transcriptional level, requiring NF-B activation and developing over 4–6 h. Ubiquitin-conjugating activity then remains elevated for at least 24 h after exposure to TNF-. This persistent response should not be confused with the transient increase in ubiquitin conjugation that occurs in muscle cells immediately upon TNF- exposure (8)
. The latter is a signaling event that stimulates I-B degradation and nuclear translocation of NF-B, then terminates within minutes. The transient response is regulated by a distinct E2 (UbcH7) and is not under transcriptional control (42)
.
UbcH2 appears to be essential for the persistent rise in ubiquitin-conjugating activity induced by TNF-. Addition of recombinant dominant negative C87S-UbcH2 inhibited ubiquitin-conjugating activity in extracts obtained from TNF--conditioned myotubes. Under our conditions, the mutant protein may compete with endogenous UbcH2 for interaction with critical ubiquitin ligase(s) or may function as a sink for 125I-ubiquitin. Similar studies were conducted using a dominant negative mutant of E214k that interacts with E3 to mediate degradation of muscle protein in a variety of catabolic states (43)
. Results indicate that E214k also contributed to the ubiquitin-conjugating activity measured in TNF--treated myotubes. This is likely to reflect activity of the constitutive enzyme since TNF- exposure did not alter E214k expression. In combination, our findings suggest that UbcH2 and E214k act in parallel, perhaps targeting distinct pools of protein for degradation. It is noteworthy that ubiquitin conjugation could be accelerated by adding either recombinant UbcH2 or recombinant E214k to whole-cell extracts. This observation indicates E2 availability was rate limiting under the conditions of our experiment. It will be important to establish whether levels of these or other E2s limit the breakdown of muscle protein in vivo.
Original descriptions of UbcH2 showed that the enzyme can catalyze ubiquitin conjugation to histone H2A in the absence of an E3 protein (19
, 35)
, a finding that we reproduced. This led Kaiser and colleagues (35)
to suggest that UbcH2 might function without an E3 partner. Subsequent research does not support this model (44)
. For example, E214k can catalyze ubiquitin conjugation to histone but is known to function primarily in concert with E3 (45)
. Likewise, in the absence of an E3, UbcH2 did not catalyze ubiquitin conjugation to any of four myofilament proteins or to lactalbumin. It is likely that UbcH2 interacts with one or more ubiquitin ligases in vivo. The most obvious candidates are those known to play an important role in skeletal muscle atrophy: E3 (43)
, atrogin/MAFbx (46
, 47)
, and MuRF1 (47)
.
In summary, we have identified UbcH2 gene transcription as a downstream target for TNF-/NF-B signaling in skeletal muscle. This leads to increased UbcH2 content and elevated ubiquitin-conjugating activity in skeletal muscle. These findings identify a novel mechanism whereby TNF- may promote the loss of muscle protein.
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ACKNOWLEDGMENTS |
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Received for publication September 16, 2002. Accepted for publication February 14, 2003.
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