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-II during Cancer Cachexia
1 Department of Metabolic Disorders, Oncology & Discovery Research, Amgen Inc., Thousand Oaks, California; and 2 Laboratory of Protein Catabolism, Department of Oncology, University of Alberta, Edmonton, Alberta, Canada
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-II, a novel "N-end rule" ubiquitin ligase, and its functional involvement in cancer cachexia. E3-II is highly enriched in skeletal muscle, and its expression is regulated by proinflammatory cytokines. In two different animal models of cancer cachexia, E3-II was significantly induced at the onset and during the progression of muscle wasting. The E3-II activation in skeletal muscle was accompanied by a sharp increase in protein ubiquitination, which could be blocked by arginine methylester, an E3-selective inhibitor. Treatment of myotubes with tumor necrosis factor or interleukin 6 elicited marked increases in E3-II but not E3-I expression and ubiquitin conjugation activity in parallel. E3-II transfection markedly accelerated ubiquitin conjugation to endogenous cellular proteins in muscle cultures. These findings show that E3-II plays an important role in muscle protein catabolism during cancer cachexia and suggest that E3-II is a potential therapeutic target for muscle wasting. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The N-end rule pathway (7)
is one of the best-characterized ubiquitin systems, which is known to selectively degrade proteins with basic or large hydrophobic NH2-terminal residues. The UBR1 gene encoding the N-end rule ubiquitin ligase has been studied intensively in yeast (7)
. A mammalian counterpart of yeast UBR1, referred to as E3, recently was reported, including a full-length mouse cDNA and a partial human cDNA (8)
. Biochemical studies suggest that the N-end rule pathway catalyzes the breakdown of a major fraction of soluble proteins in skeletal muscle (9, 10)
and is accelerated during pathologic states of muscle wasting (11)
. However, it remains unclear as to which ubiquitin ligase(s) is directly involved in the control of muscle protein catabolism.
Here, we have cloned the human and mouse full-length cDNAs encoding a novel N-end rule ubiquitin ligase, E3-II, and compared its functional significance with the reported mammalian N-end rule ubiquitin ligase UBR1/E3 (ref. 8
; referred to as E3-I herein) with respect to its role in protein catabolism during cancer cachexia. We found that E3-I and E3-II were significantly up-regulated in skeletal muscle during cancer cachexia and that transfection of either E3-I or E3-II dramatically stimulates protein ubiquitination in muscle cells. However, E3-II appeared to be more critically involved in muscle wasting because E3-II was not only more specifically expressed in muscle tissues but also it was differentially activated by tumor necrosis factor (TNF-) or interleukin 6 (IL-6), major proinflammatory cytokines known to be involved in the development of cachexia (2
, 12, 13, 14)
. Furthermore, E3-II expression was significantly up-regulated at the early onset of muscle wasting when E3-I expression was unchanged. We postulate that the novel N-end rule ubiquitin ligase E3-II is an important downstream molecular target for muscle protein catabolism and discuss future experiments to explore the therapeutic importance of the E3 family in muscle wasting.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-II, muE3-II, and huE3-I, verified through confirmation sequencing, and deposited into American Type Culture Collection (Manassas, VA).
Cell Culture and Transfection.
C2C12 and L6 myoblasts were obtained from American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented (Life Technologies, Rockville, MD) with 10% fetal bovine serum and 1x L-glutamine. Cell differentiation was induced with Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum and L-glutamine for 96 hours. Differentiated myotubes were treated with 10 ng/mL TNF- (R&D Systems, Minneapolis, MD) or with 10 ng/mL IL-6 (R&D Systems) up to 5 days. For transfection, huE3-II and huE3-I were subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) to generate pcDNA-E3-II and pcDNA-E3-I. The cultures were transiently transfected with pcDNA-E3-I (E3-I) or pcDNA-E3-II (E3-II) or mock transfected with pcDNA3.1 only (Mock) using Lipofectamine 2000 and the manufacturer’s protocols (Life Technologies).
Northern Blot Analysis.
Pair-fed, non–tumor-bearing control and tumor-bearing animals (n = 6 per mouse group; n = 8 per rat group) were killed by CO2 asphyxiation at 3 and 5 days post–Yoshida ascites hepatoma (YAH) tumor implantation (rats) and at 12 and 17 days after colon-26 adenocarcinoma (C26) tumor implantation (mice). Both the medial gastrocnemius muscles were rapidly dissected and frozen immediately in liquid nitrogen. All of the gastrocnemius muscles collected from each experimental animal group were combined. RNA was isolated from each of the pooled muscle samples and from the myotube cultures by using the TRIzol reagent following manufacturer’s protocols (Life Technologies). Equal amounts of total RNA (20 µg per lane) were separated by electrophoresis through 1% agarose gels. The separated RNA was transferred to nylon membranes and cross-linked by exposure to UV light. The membranes containing mouse or rat RNA were hybridized with cDNA probes for muE3-II or muE3-I (corresponding to amino acid position 360 to 517 and 361 to 517, respectively). Human multiple tissue RNA blots (Clontech) were hybridized with cDNA probes for huE3-I or huE3-II (corresponding to amino acid position 1157 to 1388 and 1186 to 1337, respectively). Radiolabeling of cDNA probes with [32P]dCTP was performed using the Prime-It-RmT Random Primer labeling kit (Stratagene, La Jolla, CA). Membranes were prehybridized, hybridized, and washed using the method of Church and Gilbert (15)
and exposed to X-ray film (Kodak, Rochester, NY) at –70°C. All of the probed blots subsequently were stripped and rehybridized with a [32P]dCTP-labeled ß-actin probe (Clontech) to confirm that equal amounts of RNA were transferred to the membranes. E3 expression levels were analyzed using PhosphorImager (STORM 860; Amersham Biosciences, Piscataway, NJ) equipped with quantitation software (ImageQuant 5.0; Amersham Biosciences) and were normalized against ß-actin levels.
Ubiquitin Conjugation Assays.
The assay conditions basically were the same as described previously (11)
. C2C12 or L6 cell lysates were prepared in ice-cold lysis buffer [50 mmol/L Tris-HCL (pH 8.0), 2 mmol/L DTT, and 5 mmol/L MgCl2] supplemented with a protease inhibitor mixture (Sigma Chemical Co., St. Louis, MO). The crude lysates were centrifuged at 10,000 x g for 10 minutes, and supernatants were used for ubiquitination assay. Fraction II of muscle extracts was prepared from the frozen gastrocnemius muscles of each experimental animal group (n 
6) as described previously (11)
. Ubiquitin conjugation to endogenous soluble proteins using 125I-ubiquitin (0.15 mg/mL; 
x107 cpm) and exogenous N-end rule substrate, human -lactalbumin, was carried out as described previously (10
, 11)
. Cell lysates prepared from cultures that had been treated with 10 ng/mL TNF- (3 or 4 days) or with 10 ng/mL IL-6 (3 or 5 days) and from untreated cultures (control) were used for ubiquitin conjugation reactions. When effects of an E3 inhibitor were measured, ubiquitin conjugation was carried out in the presence of the E3 inhibitor arginine methylester (ArgME) or alanine methylester (AlaME) as negative control at final concentrations of 10 mmol/L.
Cancer Cachexia Animal Models.
The YAH-130 cancer cachexia rat model and the C26 cancer cachexia mouse model and pair feeding were essentially the same as described previously (19, 20, 21, 22)
. Female Sprague Dawley rats of the Buffalo strain weighing 200 g and male 9-week-old CDF1 mice were used for the implantation experiments. Two different treatments were compared: tumor-bearing and pair-fed control animals. Rats were implanted with 100 µL of ascites fluid containing YAH-130 tumor cells from a single donor animal or with an equal volume of saline buffer. Mice were injected subcutaneously with either 0.5 x 106 C26 tumor cells or an equal volume of saline buffer.
Accession Numbers.
The GenBank accession numbers are as follows: AY061884 for huE3-II, AY061885 for muE3-II, and AY061886 for huE3-I.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-II and E3-I and Murine E3-II. DNA sequence (8)
as query, we performed extensive bioinformatic analysis against the public (GenBank) and the Amgen internal (Amgenesis) EST databases, followed by homology cloning. This led to the isolation of mouse and human full-length cDNAs encoding the entire open reading frame of a novel E3 ubiquitin ligase, E3-II (referred to as muE3-II and huE3-II, respectively). We denote it as E3-II because of its structural homology and activity resemblance to the known murine N-end rule ubiquitin ligase UBR1/E3 (referred to as muE3-I herein; ref. 8
). For structural analysis, we also isolated the full-length cDNA encoding human E3-I (referred to as huE3-I). Transformation with full-length E3-II or E3-I cDNA appeared to be toxic to Escherichia coli, and as a result, the frequencies of getting transformants containing the full-length E3-II or E3-I cDNA inserts were <1/500. We verified the sequences of the full-length cDNA inserts by confirmation sequencing against multiple clones and subcloned the confirmed full-length cDNAs into the mammalian expression vector pcDNA3.1 for transfection purposes. To our knowledge, this is the first successful isolation of full-length E3-II and the first successful attempt to construct full-length E3-II and E3-I mammalian expression vectors. To define the genomic structures of the human E3 family, we performed genomic database analysis using the full-length human E3-II and E3-I cDNAs as queries. The results revealed that the human E3-II gene consists of 47 exons and is located on chromosome 6, whereas the human E3-I gene is made up of 48 exons and is located on chromosome 15. Fig. 1
shows the sequence alignment of huE3-II, muE3-II, and huE3-I with the reported mouse E3/UBR1 (muE3-I; ref. 8
) at amino acid level. E3-II and E3-I exhibit 58% overall sequence homology and greater homology within a number of highly conserved regions, including domains I through V and the basic residue-rich region originally reported for yeast UBR1 (8
, 17)
. In addition, 41 identical cysteine residues and the residues in yeast UBR1 that were reported to be necessary for type 1 or type 2 substrate binding (7
, 8
, 17, 18)
are found to be conserved. These structural features identify E3-II as a new N-end rule ubiquitin ligase and clearly define the existence of a mammalian E3 ubiquitin ligase family.
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-II.-II– or E3-I–selective probes (see Materials and Methods) revealed that the tissue expression profile of E3-II was different from that of E3-I. E3-II is the more muscle-specific form of E3 family (Fig. 2A)
in human tissues and was found to be most highly expressed in skeletal muscle with moderate expression in heart, minor expression in kidney, and minimal expression in other tissues examined, including brain, colon, thymus, spleen, liver, intestine, placenta, lung, and peripheral WBCs. In contrast, moderate levels of E3-I (Fig. 2C)
were found to spread through many nonmuscle tissues examined, although skeletal muscle and heart had relatively higher levels of the E3-I message. Similar results were obtained from Northern blot analysis using mouse and rat tissues (data not shown). The E3-II–selective probes detected two bands in blots containing human or mouse RNA isolated from muscle tissues, including a higher molecular weight band of >7.5 kb and a lower molecular weight band of 6 kb (Fig. 2A and B)
. However, when rat tissue RNA was examined, only a single band of >7.5 kb was detected with the same E3-II–selective probes (Fig. 3B)
. Whether the lower molecular weight band seen in human and mouse Northern blots corresponds to an alternatively spliced form of E3-II or a homologous gene needs to be further investigated.
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Family in Muscle Protein Catabolism during Cancer Cachexia. gene family in muscle protein catabolism during disease states, we analyzed the expression levels of E3-II and E3-I in skeletal muscle using two tumor-implantation models of cancer cachexia: YAH-130 tumor-bearing rats (19
, 22) and C26 tumor-bearing mice (20
, 21)
. These tumor-bearing models mimic human cancer cachexia with a pronounced loss of muscle mass at low tumor burden. The tumor-bearing animals also show a reduction in food intake (19, 20, 21, 22)
. However, the rapid muscle wasting could not be accounted for by reduced food intake because pair feeding that simulated the semifasting condition in the tumor-bearing state had no significant effect on muscle mass. It has been shown that in the YAH model, the rate of muscle protein catabolism increases rapidly in a manner that is independent of food intake by 63% to 90% at 5 days of tumor implantation (19
, 22)
. Detailed descriptions of food intake, progressive weight loss, and loss of muscle protein mass and selective activation of ubiquitin-proteasome protein catabolism are provided elsewhere (19
, 22)
. Under pair-feeding conditions, a progressive loss of skeletal muscle weight was observed ranging from 4% to 9% decrease at the early stage (12 days post-C26 implantation and 3 days post–YAH-130 implantation) to 25% to 26% decrease at the late stage (17 days post-C26 tumor implantation and 5 days post–YAH-130 tumor implantation) relative to the non–tumor-bearing paired-fed control animals (Fig. 3A)
. We examined in parallel the expression levels of E3-I and E3-II and the ubiquitin-protein conjugation activities in skeletal muscle at the early and late stages of muscle wasting in these models. Changes in E3-II occurred concordantly with the early onset and the continued progression of muscle atrophy in both models examined. As shown in Fig. 3
, at the early stage of muscle wasting, there was a significant (approximately twofold) increase in E3-II expression in the gastrocnemius muscle of the tumor-bearing animals as compared with that in pair-fed control animals, whereas the level of E3-I appeared to be unaltered (Fig. 3B)
. At the later stage of muscle wasting, increased expression of E3-II (more than threefold) and E3-I (twofold to threefold) was detected in the gastrocnemius muscle of the tumor-bearing animals as compared with their expression levels in pair-fed, non–tumor-bearing control animals (Fig. 3B)
. Moderate levels of E3-I and E3-II were expressed in heart; however, Northern blot analysis revealed no significant alteration in either E3-I or E3-II expression in heart at the early and late stages of tumor implantation of mice bearing C26 (data not shown). To determine the rates of ubiquitin conjugation in skeletal muscle from the animal models, muscle lysates (fraction II) of gastrocnemius muscles from tumor-bearing and pair-fed control animals were subjected to ubiquitination reactions in the presence of 125I-ubiquitin (see Materials and Methods). In parallel with the increase in E3-II expression at the early stage, as well as with the increases in E3-II and E3-I expression at the late stage, there was a significant increase in ubiquitin conjugation activities in the gastrocnemius muscle of the YAH-130 tumor-bearing rats (Fig. 3C)
and C26 tumor-bearing mice (data not shown). We also examined whether the activation of the N-end rule pathway mediated the accelerated muscle protein ubiquitination. Ubiquitin conjugation assays revealed that the gastrocnemius muscle lysates from the YAH-130 tumor-bearing rats (Fig. 3C)
and the C26 tumor-bearing mice (data not shown) catalyzed the ubiquitination of exogenously added 125I–-lactalbumin, a bona fide N-end rule substrate, significantly faster than those from pair-fed control animals. Moreover, the addition of the E3-selective inhibitor ArgME (7
, 10 , 18)
to the muscle lysates virtually abolished the accelerated ubiquitination activity seen in the cachectic muscle lysates (Fig. 3C)
. In a separate study using recombinantly produced E3-II, we confirmed that E3-II was capable of catalyzing ubiquitin conjugation to -lactalbumin and highly susceptible to ArgME inhibition.3
These results indicate that the increased ubiquitination activities in the atrophying muscle were attributable to the activation of the E3 family. Collectively, our findings show that the transcriptional activation of E3-II, in particular at the onset of wasting, as well as the transcriptional coactivation of E3-II and E3-I as cachexia progresses, may contribute to accelerated protein breakdown and muscle wasting. This will be further clarified when gene deletion mutants or selective small molecule inhibitors can be analyzed.
Differential Induction of E3-II by TNF- and IL-6.
Using differentiated myotube cultures, we examined whether TNF- and IL-6 were capable of activating the E3 family in muscle cells because these two major proinflammatory cytokines were known to be the key humoral mediators of muscle wasting and cachexia in the C26 and YAH-130 tumor-implantation models used in our studies. IL-6 was reported as a cachectic factor in the development of cancer cachexia in the C26 model (20
, 21)
, whereas TNF- was shown to mediate the activation the ubiquitin-dependent proteolytic system in the YAH model (23)
. Fig. 4A
shows the results of Northern blot analysis of E3-II and E3-I levels in differentiated C2C12 myotube cultures that had been incubated with or without treatment with TNF- or IL-6. Remarkably, treatment with either TNF- or IL-6 resulted in a twofold to fourfold induction in E3-II expression without detectable alteration in E3-I expression. Parallel examination of the ubiquitination activities in lysates of TNF-– or IL-6–treated cultures revealed that TNF- or IL-6 treatment led to a significant increase in the ubiquitin conjugation to endogenous cellular proteins (Fig. 4B
, left) and to exogenously added -lactalbumin (Fig. 4B
, right). These data show that TNF- and IL-6 stimulate protein ubiquitination in muscle cells via, at least in part, the induction of E3-II. Many proinflammatory cytokines, including TNF- and IL-6, have been shown to be involved in human cachectic disease states, such as cancer cachexia, AIDS, inflammatory cachexia, renal cachexia, burns, and sepsis (1, 2, 3
, 23, 24, 25, 26, 27, 28)
. E3-II activation in muscle may be an important molecular mechanism by which proinflammatory cytokines and possibly other cachectic factors induce protein catabolism and muscle wasting.
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-I and E3-II on Protein Ubiquitination in Muscle Cell Cultures. gene expression leads to accelerated ubiquitination in muscle cells, we transfected mouse C2C12 and rat L6 myotube cultures with huE3-II and huE3-I and performed ubiquitin conjugation assays using freshly prepared cell lysates. Transfection of C2C12 cultures with either huE3-II or huE3-I dramatically stimulated the ubiquitination of the endogenous cellular proteins (Fig. 4C
, left). Similar results were obtained from E3-II– or E3-I–transfected L6 myotube cultures (data not shown). Lysates from E3-II– or E3-I–transfected cultures catalyzed ubiquitin conjugation to exogenously added 125I–-lactalbumin significantly faster than those from control cultures, whereas addition of ArgME virtually completed blocked this accelerated ubiquitin conjugation activity (Fig. 4C
, right). Therefore, an increase in the expression of either E3-II or E3-I is sufficient to cause accelerated protein ubiquitination in muscle cells. Ubiquitin conjugation is the prerequisite and rate-limiting step in proteasome-mediated proteolysis. Our results are consistent with the report that the N-end rule pathway activity mediates the breakdown of a major fraction of muscle proteins (9
, 10)
and further pinpoint a pivotal role of E3-II and E3-I, rate-limiting enzymes of the pathway, in the regulation of muscle protein catabolism. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-II and elucidated its critical role in mediating muscle protein ubiquitination and in particular its regulation during cancer cachexia and by proinflammatory cytokines. Our data show that E3-II plays a key role in muscle protein catabolism in the two experimental models of cancer cachexia examined. At the early onset of muscle wasting in the C26 and YAH-130 tumor-implantation models, a differential induction of E3-II, but not E3-I, occurred concomitantly with a significant increase in ubiquitin conjugation to endogenous muscle proteins and to N-end rule model substrate -lactalbumin. At this early stage of muscle wasting, addition of E3-selective inhibitor ArgME completely abolished the increased ubiquitination activity, indicating that the activation of E3-II was responsible for the accelerated ubiquitin conjugation activity. That overexpression of E3-II leads to accelerated protein ubiquitination has been unequivocally shown with our E3-II transfection experiments. Similar to what was observed in cancer cachexia models, transfected muscle cultures show a sharp increase in ubiquitin conjugation to endogenous proteins and to exogenously added N-end rule model substrate. It is noteworthy that E3-II is not only muscle specific but also the proinflammatory cytokine-inducible form of the E3 family. TNF- and IL-6 treatment differentially induced the expression of E3-II and a parallel increase in ubiquitin conjugation activity in cultured myotubes without affecting E3-I expression levels. We also examined MAFbx/Atrogin-1 expression in myotube cultures treated with TNF- or IL-6 but observed no significant change in its expression (data not shown). Our in vitro results corroborate our in vivo findings in YAH and C26 cachexia models, in which TNF- and IL-6 were known to play a key role in protein catabolism and the development of muscle wasting. Two other ubiquitin ligases, including MuRF1 (29) , a RING finger protein, and MAFbx or Atrogin-1 (29 , 30) of the SCF family, recently also have been reported to play a role in muscle atrophy. Conceivably, multiple ubiquitin ligases may operate in muscle atrophy by different mechanisms, with each playing a nonredundant role. Additional experiments will be needed to clarify the relative contribution of different ubiquitin ligases to muscle wasting under different disease conditions.
Further studies are required to better understand the importance of the E3 ubiquitin ligase family in catabolic disease states. These include identifying the physiologic substrates for E3-II and E3-I in skeletal muscle, elucidating various signaling events that regulate the activity of the E3 family, and analyzing the effects of E3 blockade through gene ablation and/or the design of selective small molecule inhibitors on animal’s tolerance to cachectic challenges. Ubiquitin ligases are attractive molecular targets for manipulation of proteolysis because they are muscle-specific isoforms, and their activation may be specific to different forms of muscle wasting, such as disuse atrophy or that associated with cancer and inflammation. These features may potentially allow for local suppression of muscle catabolism without affecting the basal proteolytic processes in nonmuscle tissues or associated with essential functions, such as antigen processing in antigen-presenting cells. Activation of the ubiquitin-proteasome system is common to many models of cancer cachexia regardless of whether one or another hormone, cytokine, or other factors appear to be the humoral signal for the system’s activation (31)
. The position of E3 in the span of the pathway of muscle protein catabolism that is common to multiple hormones, cytokines, and other factors would allow for a simplification of anticatabolic therapies directed at this step rather than attempts to individually monitor and manipulate humoral mediators of diverse types in cancer patients.
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ACKNOWLEDGMENTS |
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-II purification, and ubiquitination assay. |
FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Notes: W. J. Boyle is currently at Auxeris Therapeutics, St. Louis, Missouri.
Requests for reprints: H. Q. Han, Department of Metabolic Disorders, Amgen Research, One Amgen Center Drive, Thousand Oaks, CA 91320. Phone: 805-447-4770; E-mail: hqhan{at}amgen.com
Received 6/15/04. Revised 8/12/04. Accepted 9/14/04.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Nat Biotechnol 1999;17:666-9.[CrossRef][Medline]
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