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Biochemical and Biophysical Research Communications 267, 504–508 (2000)

doi:10.1006/bbrc.1999.1987, available online at http://www.idealibrary.com on

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he Gene Expression of Ubiquitin Ligase E3as Upregulated in Skeletal Muscle during Sepsisn Rats—Potential Role of Glucocorticoids

avid Fischer, Xiaoyan Sun, Gyu Gang, Tim Pritts, and Per-Olof Hasselgrenepartment of Surgery and Department of Molecular and Cellular Physiology, University of Cincinnati,incinnati, Ohio 45267; and Shriners Hospital for Children, Cincinnati, Ohio

eceived November 17, 1999

dependent mechanisms (6–8). In recent studies wefarpo(d

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Muscle protein breakdown during sepsis is associ-ted with upregulated expression and activity of thebiquitin-proteasome proteolytic pathway. Previoustudies suggest that ubiquitination of proteins in skel-tal muscle is regulated by the ubiquitin ligase E3aogether with the 14 kDa ubiquitin-conjugating en-yme E214k. The E3a gene was cloned only recently.he influence of sepsis on the gene expression of E3a

n skeletal muscle has not been reported. In theresent study, induction of sepsis in rats by cecal liga-ion and puncture resulted in increased mRNA levelsor E3a in white, fast-twitch but not in red slow-twitch

uscle. Treatment with the glucocorticoid receptorntagonist RU38486 (10 mg/kg) prevented the sepsis-nduced increase in E3a and E214k mRNA levels. Theresent study is the first report of increased E3a ex-ression in skeletal muscle during sepsis. The results

end further support to the concept that glucocor-icoid-mediated upregulation of the ubiquitin-protea-ome proteolytic pathway is involved in sepsis-in-uced muscle cachexia. Increased expression of both3a and E214k suggests that muscle proteins are de-raded in the N-end rule pathway during sepsis. © 2000

cademic Press

Muscle cachexia during sepsis and after severe in-ury is mainly caused by increased protein breakdown,n particular myofibrillar protein breakdown (1, 2).keletal muscle is the most important source of nitro-en loss in septic and injured patients (3). Musclereakdown results in muscle weakness and fatigue andncreases the risks for pulmonary and thromboembolicomplications (4, 5). A better understanding of theegulation of protein breakdown in cachectic muscleherefore has important clinical implications.

Intracellular protein degradation is regulated byultiple proteolytic pathways, including lysosomal,

alcium-calpain-mediated and ubiquitin-proteasome-

504006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

ound evidence that muscle breakdown during sepsisnd after severe burn injury was associated with up-egulated expression and activity of the ubiquitin-roteasome pathway (2, 9, 10), similar to a number ofther catabolic conditions such as cancer (11, 12), AIDS13), denervation (14), fasting (15) and metabolic aci-osis caused by kidney failure (16).In the ubiquitin-proteasome pathway, proteins that

re to be degraded are first conjugated to multipleolecules of ubiquitin whereafter they are recognized

nd degraded by the 26S proteasome (6, 17). The ubiq-itination process is regulated by three sets of en-ymes: the ubiquitin activating enzyme E1; the ubiq-itin-conjugating enzyme E2; and ubiquitin ligase E3.he ubiquitin system is hierarchal in that there is one1 with multiple E2s and E3s. To date, more than 30biquitin-conjugating enzymes and 25 ubiquitin li-ases have been identified. Individual E3s account forubstrate specificity and also act in concert with spe-ific E2s (17).The ubiquitin ligase E3a regulates the ubiquitina-

ion of proteins in the so called N-end rule pathway18). Proteins degraded in this pathway have a desta-ilizing N-end consisting of one of the basic aminocids Arg, Lys or His or one of the bulky hydrophobicmino acids Phe, Leu, Trp, Tyr or Ile and an internalys to which ubiquitin is conjugated by the 14 kDabiquitin-conjugating enzyme E214k (18). Previous re-orts by Solomon et al. (19, 20) suggest that a largeroportion of muscle proteins are degraded in the-end rule pathway and that this pathway may ac-

ount for stimulated muscle proteolysis in cancer andepsis. We recently found that the gene expression of214k was upregulated in septic muscle, further sup-orting a role of this pathway in sepsis-induced muscleachexia (21).The influence of sepsis on the gene expression of E3a

as not been reported. The recent cloning of the E3a

gene by Varshavsky and co-workers (22) has made itpoteec2isR

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Vol. 267, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ossible to examine the effects of different conditionsn the expression of E3a. The purpose of this study waso test the hypothesis that sepsis upregulates the genexpression of E3a in skeletal muscle. Because in otherxperiments we have found evidence that glucocorti-oids regulate protein breakdown in septic muscle (23,4), we also tested the role of glucocorticoids in sepsis-nduced changes in muscle E3a expression by treatingeptic rats with the glucocorticoid receptor antagonistU38486 (25).

ATERIALS AND METHODS

Experimental animals. Sepsis was induced in male Sprague-awley rats (40–60 g) by cecal ligation and puncture (CLP) asescribed previously (1, 2). Control rats underwent sham-operation,.e., laparotomy and manipulation but no ligation or puncture of theecum. All rats were resuscitated with 100 ml/kg body weight ofaline administered subcutaneously on the back at the time of sur-ery. The rats had free access to drinking water but food was with-eld after surgery in order to avoid any influence on metabolichanges caused by different food intake between the two groups ofats. At different time points up to 16 h after CLP or sham-operation,he extensor digitorum longus (EDL) and soleus muscles were har-ested and frozen at 270°C for subsequent study. In previous re-orts, total and myofibrillar ubiquitin-dependent muscle proteinreakdown was increased and rats were in a hyperdynamic state ofepsis 16 h after CLP (2).The experimental model of sepsis used here is clinically relevant

ecause it resembles the situation in patients with sepsis caused byntraabdominal abscess and devitalized tissue. In several previouseports from our laboratory, CLP in rats resulted in upregulatedbiquitin-proteasome-dependent muscle proteolysis (2, 24, 26, 27).n those studies, rats weighing 40–60 g were used in order to maket possible to measure protein turnover rates in incubated lowerxtremity muscles. Rats of the same size were used here to allow foromparisons with results in our previous reports. It should be notedhat although protein turnover rates are higher in young growingats than in adult rats, the relative changes in muscle protein turn-ver induced by sepsis are similar in young and adult rats (28). Inddition, we recently found that the metabolic response to sepsis inkeletal muscle, including upregulated expression of the ubiquitin-roteasome pathway, was similar in adult patients and young grow-ng rats (9), further validating the use of small rats in the study ofepsis-induced muscle cachexia.In order to study the role of glucocorticoids in sepsis-related

hanges in muscle mRNA levels, groups of rats were treated with thelucocorticoid receptor antagonist RU38486 (Research Biochemicalsnternational, Natick, MA). The drug was administered by gavageh before sham-operation or CLP at a dose of 10 mg/kg in a suspen-

ion containing 0.25% carboxymethylcellulose and 0.20% polysor-ate. Other rats received a corresponding volume (0.5 ml/100 g) ofehicle by gavage 2 h before sham-operation or CLP. RU38486 is aotent glucocorticoid receptor antagonist that has no agonist activityven at high concentrations (25). The drug was used in previoustudies in our laboratory and prevented sepsis-induced changesn muscle protein breakdown and expression of the ubiquitin-roteasome proteolytic pathway (23, 24). Sixteen hours after sham-peration or CLP, EDL muscles were harvested and frozen at 270°Cor subsequent study.

All experiments were conducted and animals were cared for inccordance with the National Research Council’s Guide For the Carend Use of Laboratory Animals. The experimental protocols werepproved by the Institutional Animal Care and Use Committee athe University of Cincinnati.

505

inium thiocyanate-phenol-chloroform extraction as described byhomczynski and Sacchi (29) using an RNA STAT-60 kit (Tel-Test

B,” Friendswood, TX). RNA was denatured and separated by elec-rophoresis on 0.8% agarose gel containing formaldehyde. The RNAas transferred from the gel to nylon membranes (Micron Separa-

ions, Westboro, MA) by capillary action in 203 SSC (1 SSC 5 0.15NaCl, 15 mM sodium citrate) overnight. RNA was immobilized by

aking at 80°C for 1 h. The blots were prehybridized at 42°C for 4 hn 50% formaldehyde, 63 SSPE (13 SSPE 5 0.15 M NaCl, 10 mMaH2PO4, 1 M EDTA), 53 Denhardt’s solution, 0.5% SDS, and 100g/ml salmon sperm DNA. cDNA probes were labeled by randomriming with [32P]dCTP (Stratagene, La Jolla, CA). The blots wereybridized with the 32P-labeled cDNA probe in the same buffer asescribed above at 42°C overnight. The blots were then washed twicen 13 SSPE and 0.1% SDS at room temperature, washed once in.13 SSPE and 0.1% SDS at 45°C, and exposed to phosphor screenor quantitation of signal intensity in a Phosphoimager SF (Molecu-ar Dynamics, Sunnyvale, CA). The membranes were stripped andehybridized with a rat GAPDH cDNA probe and processed as above,nd the mRNA abundance was expressed as the ratio to GAPDHRNA. The E214k and GAPDH cDNA probes were generated as

escribed previously from our laboratory (2, 9, 21, 24) on the basis ofhe reported structure of the genes (30). The E3a cDNA probe wasynthesized by reverse transcriptase polymerase chain reaction (RT-CR) using a RT-PCR kit (Perkin Elmer Cetus, Norwalk, CT) and ratkeletal muscle total RNA. The primers were: sense, 59-GAT CCCAC AAG TTC TTG TTA CTG-39; antisense 59-CAG TTT CAT TATTT CGT TCT CAG-39 (22).

Statistics. Results are presented as means 6 SEM. Student’s-test or analysis of variance (ANOVA) followed by Tukey’s test wassed as appropriate.

ESULTS

In previous studies we found that the catabolic re-ponse to sepsis was particularly pronounced in white,ast-twitch muscle with only minor or no changes oc-urring in red, slow-twitch muscle (1, 2). In order toxamine whether sepsis results in a similar differentialesponse with regards to the expression of E3a, mRNAevels for E3a were measured in the white, fast-twitchDL and the red, slow-twitch soleus muscle 16 h afterham-operation or CLP. Sepsis resulted in an approx-mately two-fold increase in E3a mRNA in EDL but didot influence E3a mRNA levels in soleus muscle (Fig. 1).In order to examine how soon after the onset of

epsis the gene expression of E3a is upregulated,RNA levels were measured in EDL muscles at differ-

nt time points after sham-operation or CLP. E3aRNA levels were significantly elevated 8 h and 16 h

fter induction of sepsis whereas at 4 h, no significantifference between sham-operated and septic rats waseen (Fig. 2).Results in previous reports suggest that glucocorti-

oids are the most important factor regulating the cat-bolic response in skeletal muscle (24, 31). We nextested the role of glucocorticoids in sepsis-induced in-rease in E3a mRNA levels by treating rats with thelucocorticoid receptor antagonist RU38486. Whenats were treated with 10 mg/kg of RU38486 2 h beforeham-operation or CLP, the sepsis-induced increase in

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Vol. 267, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3a mRNA levels was significantly reduced (Fig. 3),uggesting that glucocorticoids participate in the reg-lation of E3a in skeletal muscle during sepsis.E3a is important for the ubiquitination of proteins in

he N-end rule pathway (18). The ubiquitin-conju-ating enzyme E214k acts in concert with E3a in thebiquitination of proteins in this pathway. We recentlyeported that sepsis upregulates the gene expression of214k in skeletal muscle (21) but the role of glucocorti-

oids in the regulation of E214k during sepsis is notnown. We therefore tested the effect of RU38486 onepsis-induced changes in E214k mRNA levels in EDLuscle. Similar to our previous report (21), sepsis re-

ulted in increased mRNA levels for the 1.2 kb tran-cript of the E214k gene (Fig. 4) with no changes seen inhe levels of the 1.8 k transcript (not shown). Theepsis-induced increase in E214k 1.2 kb mRNA levelsas blocked by treatment with RU38486 (Fig. 4) sug-esting that the gene expression of E214k in skeletaluscle is regulated by glucocorticoids during sepsis.

ISCUSSION

In the present study, sepsis resulted in increasedene expression of the ubiquitin ligase E3a in skeletaluscle and because this response to sepsis was blunted

n rats treated with RU38486, it is likely that upregu-

FIG. 1. Effect of sepsis, induced by cecal ligation and punctureCLP) in rats, on E3a mRNA in EDL and soleus muscles. Represen-ative Northern blots are shown in the upper panel and quantitationf mRNA levels from 5 sham-operated (open bars) and 5 septic (filledars) rats is shown in the lower panel. *P , 0.05 vs sham EDL bytudent’s t-test.

506

art regulated by glucocorticoids. The E3a gene wasloned only recently (22) and to our knowledge theresent report is the first study to demonstrate in-reased gene expression of E3a in skeletal muscle dur-ng sepsis.

The present finding of increased expression of E3aogether with our previous report of upregulated ex-ression of E214k in septic muscle (21) support theoncept that degradation of muscle proteins occur inhe N-end rule pathway during sepsis. The currenttudy did not address the question which proteins thatre degraded in this pathway during sepsis but othereports from our laboratory provided evidence that it isainly the myofibrillar proteins actin and myosin that

re broken down in septic muscle (1). In recent exper-ments, we found that myofilaments (actin and myosin)re released from the myofibrils by a calcium-calpain-ediated mechanism in skeletal muscle during sepsis

32) and it may be speculated that the released myo-laments have N-end properties that make them sus-eptible to ubiquitination by E214k and E3a.The selective increase noted here in the expression of3a in white, fast-twitch skeletal muscle is similar tohanges in total and myofibrillar protein breakdownates and to upregulation of the gene expression forbiquitin and several of the 20S proteasome subunitsuring sepsis (1, 26). Although the reason for the dif-

FIG. 2. Changes in E3a mRNA levels in rat EDL muscles atifferent time points after induction of sepsis by CLP. Representa-ive Northern blots are shown in the upper panel, and the increase inRNA levels in septic rats compared to sham-operated rats at dif-

erent time points is shown in the lower panel. The data in the loweranel are based on 5 sham-operated and 5 septic rats at each timeoint. *P , 0.05 vs sham at corresponding time point.

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erence in sensitivity between different types of skele-al muscle is not known, a similar preferential increasen protein degradation in fast-twitch muscle was notedn other catabolic conditions as well, including cancer11), fasting (15), denervation (14), glucocorticoid treat-ent (33) and burn injury (34).The catabolic response in skeletal muscle during

epsis is regulated by multiple factors, includingnterleukin-1, tumor necrosis factor (TNF) and glu-ocorticoids (23, 24, 35, 36). Among these regulators,lucocorticoids are the most important factor (31) andhere is evidence that the catabolic effects of TNF areediated by glucocorticoids as well (37). The reducedRNA levels for both E214k and E3a in septic rats

reated with RU38486 are novel observations and fur-her support the important role of glucocorticoids inhe regulation of the catabolic response to sepsis inkeletal muscle.Although the present finding of increased gene ex-

ression of E3a may be consistent with a role of thisnzyme and of the N-end rule pathway in the ubiquiti-ation and breakdown of proteins in septic muscle, theesults need to be interpreted with caution for severaleasons. First, increased mRNA levels for E3a do notecessarily mean that gene transcription was upregu-

ated but the result could also reflect increased mRNAtability. In previous studies from our laboratory, in-

FIG. 3. E3a mRNA levels in EDL muscles from sham-operatednd septic rats (CLP). Groups of rats were treated with the glucocor-icoid receptor antagonist RU38486 (10 mg/kg) or correspondingolume of vehicle administered by gavage 2 h before sham-operationr CLP. Representative Northern blots are shown in the upper panelnd quantitation of mRNA is shown in the lower panel. Open bars,ehicle; filled bars, RU38486. N 5 5 or 6 in each group. *P , 0.05 vsll other groups by ANOVA.

507

eflect increased stability of the transcripts, providingndirect evidence that gene transcription was upregu-ated (21, 26). It is possible, therefore, that the in-reased E3a mRNA levels noted here reflected in-reased transcription of the E3a gene although furtherxperiments are needed to test that notion.Second, mRNA levels may be elevated without an

ncrease in protein synthesis or protein levels. Indeed,n our previous study in which E214k mRNA levels werencreased in muscle during sepsis, E214k protein levelsere unchanged (21). Third, and perhaps most impor-

ant, increased mRNA levels for E3a do not provideny information regarding the activity of the enzyme.n recent studies by Solomon et al. (19, 20), specific E3alockers were used in a cell free system to examine theole of E3a in the ubiquitination and degradation ofuscle proteins and results from those experimentsere consistent with the concept that E3a-dependentbiquitination and degradation of proteins are in-reased in cachectic muscle. Thus, taken together, theresent results and those reported by Solomon et al.19, 20) suggest that sepsis results in transcriptionalpregulation and increased activity of E3a in skeletaluscle. The results in the present study are important

ecause a better understanding of the molecular regu-ation of muscle proteolysis may make it possible in theuture to more specifically target the catabolic responsen skeletal muscle during sepsis and perhaps other

uscle wasting conditions as well.

FIG. 4. E214k mRNA levels (1.2 kb transcript) in EDL musclesrom sham-operated and septic rats treated by gavage with RU38486filled bars) or vehicle (open bars). The groups of rats and symbols aredentical to those in Fig. 3.

ACKNOWLEDGMENTS

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18. Varshavsky, A. (1997) Genes to Cells 2, 13–28.1

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Work was supported in part by NIH Grant DK37908 and by arant from the Shriners of North America. D.F. was supported by aesearch fellowship from the Shriners of North America and G.G.nd T.P. by NIH Training Grant 1T32GM08478.

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