Recent progress in elucidating signalling proteolytic pathways in muscle wasting: potential clinical implications.

AIMS: Muscle wasting prevails with disuse (bedrest and immobilisation) and is associated with many diseases (cancer, sepsis, diabetes, kidney failure, trauma, etc.). This results first in prolonged hospitalisation with associated high health-care costs and second and ultimately in increased morbidity and mortality. The precise characterisation of the signalling pathways leading to muscle atrophy is therefore particularly relevant in clinical settings. DATA SYNTHESIS: Recent major papers have identified highly complex intricate pathways of signalling molecules, which induce the transcription of the muscle-specific ubiquitin protein ligases MAFbx/Atrogin-1 and MuRF1 that are overexpressed in nearly all muscle wasting diseases. These signalling pathways have been targeted with success in animal models of muscle wasting. In particular, these findings have revealed a finely tuned crosstalk between both anabolic and catabolic processes. CONCLUSIONS: Whether or not such strategies may be useful for blocking or at least limiting muscle wasting in weight losing and cachectic patients is becoming nowadays a very exciting clinical challenge.

Coordinate expression of the 19S regulatory complex and evidence for ubiquitin-dependent telethonin degradation in the unloaded soleus muscle.

Catabolic stimuli induce a coordinate expression of the 20S proteasome subunits in skeletal muscles. However, contradictory data have been obtained for the 19S regulatory complex (RC) subunits, which could reflect differential regulation at the transcriptional and/or translational level. To address this point we used a well-established model of muscle atrophy (hindlimb suspension) and determined the mRNA levels for 19S subunits belonging to both the base (non-ATPase S1, ATPases S7 and S8) and the lid (S14) of the 19S RC. Concomitant increased mRNA levels were observed for all studied subunits in rat soleus muscles after 9 days of unloading. In addition, analysis of polysome profiles showed a similar proportion of actively translated mRNA (50%) in unloaded and control soleus muscle. Furthermore, the repressed pool of messenger ribonucleoparticles (mRNPs) was low in both control (14%) and unloaded (15%) animals. Our data show that representative 19S subunits (S7 and S8) were efficiently translated, suggesting a coordinate production of 19S RC subunits. The 19S RC is responsible for the binding of polyubiquitin conjugates that are subsequently degraded inside the 20S proteasome core particle. We observed that soleus muscle atrophy was accompanied by an accumulation of ubiquitin conjugates. Purification of ubiquitin conjugates using the S5a 19S subunit followed by deubiquitination identified telethonin as a 26S proteasome substrate. In conclusion, muscle atrophy induces a concomitant expression of 26S proteasome subunits. Substrates to be degraded include a protein required for maintaining the structural integrity of sarcomeres.

Sensitivity and protein turnover response to glucocorticoids are different in skeletal muscle from adult and old rats. Lack of regulation of the ubiquitin-proteasome proteolytic pathway in aging.

We studied glucocorticoid-induced muscle wasting and subsequent recovery in adult (7-mo-old) and old (22-mo-old) rats, since the increased incidence of various disease states may result in glucocorticoids hypersecretion in aging. Adult and old rats received dexamethasone in their drinking water and were then allowed to recover. Muscle wasting occurred more rapidly in old rats and the recovery of muscle mass was impaired, suggesting that glucocorticoids may be involved in the emergence of muscle atrophy with advancing age. According to measurements in incubated epitrochlearis muscles, dexamethasone-induced muscle wasting mainly resulted from increased protein breakdown in the adult, but from depressed protein synthesis in the aged animal. Increased expression of cathepsin D, m-calpain, and ubiquitin was observed in the muscles from both dexamethasone-treated adult and old rats. By contrast, the disappearance of the stimulatory effect of glucocorticoids on protein break-down in aging occurred along with a loss of ability of steroids to enhance the expression of the 14-kD ubiquitin carrier protein E2, which is involved in protein substrates ubiquitinylation, and of subunits of the 20 S proteasome (the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates). Thus, if glucocorticoids play any role in the progressive muscle atrophy seen in aging, this is unlikely to result from an activation of the ubiquitin-proteasome proteolytic pathway.

Muscle wasting in a rat model of long-lasting sepsis results from the activation of lysosomal, Ca2+ -activated, and ubiquitin-proteasome proteolytic pathways.

We studied the alterations in skeletal muscle protein breakdown in long lasting sepsis using a rat model that reproduces a sustained and reversible catabolic state, as observed in humans. Rats were injected intravenously with live Escherichia coli; control rats were pair-fed to the intake of infected rats. Rats were studied in an acute septic phase (day 2 postinfection), in a chronic septic phase (day 6), and in a late septic phase (day 10). The importance of the lysosomal, Ca2+ -dependent, and ubiquitin-proteasome proteolytic processes was investigated using proteolytic inhibitors in incubated epitrochlearis muscles and by measuring mRNA levels for critical components of these pathways. Protein breakdown was elevated during the acute and chronic septic phases (when significant muscle wasting occurred) and returned to control values in the late septic phase (when wasting was stopped). A nonlysosomal and Ca2+ -independent process accounted for the enhanced proteolysis, and only mRNA levels for ubiquitin and subunits of the 20 S proteasome, the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates, paralleled the increased and decreased rates of proteolysis throughout. However, increased mRNA levels for the 14-kD ubiquitin conjugating enzyme E2, involved in substrate ubiquitylation, and for cathepsin B and m-calpain were observed in chronic sepsis. These data clearly support a major role for the ubiquitin-proteasome dependent proteolytic process during sepsis but also suggest that the activation of lysosomal and Ca2+ -dependent proteolysis may be important in the chronic phase.

Increased ATP-ubiquitin-dependent proteolysis in skeletal muscles of tumor-bearing rats.

Little information is available on proteolytic pathways responsible for muscle wasting in cancer cachexia. Experiments were carried out in young rats to demonstrate whether a small (< 0.3% body weight) tumor may activate the lysosomal, Ca(2+)-dependent, and/or ATP-ubiquitin-dependent proteolytic pathway(s) in skeletal muscle. Five days after tumor implantation, protein mass of extensor digitorum longus and tibialis anterior muscles close to a Yoshida sarcoma was significantly reduced compared to the contralateral muscles. According to in vitro measurements, protein loss totally resulted from increased proteolysis and not from depressed protein synthesis. Inhibitors of lysosomal and Ca(2+)-dependent proteases did not attenuate increased rates of proteolysis in the atrophying extensor digitorum longus. Accordingly, cathepsin B and B+L activities, and mRNA levels for cathepsin B were unchanged. By contrast, ATP depletion almost totally suppressed the increased protein breakdown. Furthermore, mRNA levels for ubiquitin, 14 kDa ubiquitin carrier protein E2, and the C8 or C9 proteasome subunits increased in the atrophying muscles. Similar adaptations occurred in the muscles from cachectic animals 12 days after tumor implantation. These data strongly suggest that the activation of the ATP-ubiquitin-dependent proteolytic pathway is mainly responsible for muscle atrophy in Yoshida sarcoma-bearing rats.

Glucocorticoids do not regulate the expression of proteolytic genes in skeletal muscle from Cushing's syndrome patients.

Glucocorticoids signal enhanced proteolysis in various instances of muscle atrophy and increased gene expression of components of the lysosomal, Ca(2+)-dependent, and/or ubiquitin-proteasome proteolytic pathways in both rat skeletal muscle and myotubes. Cushing's syndrome is characterized by chronic excessive glucocorticoid production, which results in muscle wasting. We report here no change in messenger RNA levels for cathepsin D (a lysosomal proteinase), m-calpain (a Ca(2+)-activated proteinase), ubiquitin, 14-kDa ubiquitin-activating enzyme E2, and 20S proteasome subunits (i.e. critical components of the ubiquitin-proteasome proteolytic process) in skeletal muscle from such patients. Thus, in striking contrast with animal studies, glucocorticoids did not regulate the expression of muscle proteolytic genes in Cushing's syndrome. In humans, messenger RNA levels, for at least ubiquitin and proteasome subunits, are elevated in acute situations of muscle wasting, such as head trauma or sepsis. Because Cushing's syndrome is a chronic catabolic condition, we suggest that the lack of regulation of proteolytic genes in such patients may represent an adaptive regulatory mechanisms, preventing sustained increased protein breakdown and avoiding rapid muscle wasting.

No alteration in gene expression of components of the ubiquitin-proteasome proteolytic pathway in dystrophin-deficient muscles.

Increased expression of critical components of the ubiquitin-dependent proteolytic pathway occurs in any muscle wasting condition so far studied in rodents where proteolysis rises. We have recently reported similar adaptations in head trauma patients [Mansoor et al. (1996) Proc. Natl. Acad. Sci. USA 93, 2714-2718]. We demonstrate here that the increased muscle protein breakdown seen in mdx mice only correlated with enhanced expression of m-calpain, a Ca(2+)-activated proteinase. By contrast, no change in mRNA levels for components of the ubiquitin-proteasome proteolytic process was seen in muscles from both mdx mice and Duchenne muscular dystrophy patients. Thus, gene expression of components of this pathway is not regulated in the chronic wasting that characterizes muscular dystrophy.

Nutritional and hormonal control of protein breakdown.

Multiple lines of evidence suggest that the ubiquitin-proteasome-dependent proteolytic pathway is the major degradative process responsible for the loss of muscle proteins seen in various pathological states and following food deprivation. The first step in this pathway is the covalent attachment of polyubiquitin chains to protein substrates. This signal targets the substrates for subsequent hydrolysis into peptides by the 26S proteasome. Several metabolic abnormalities (reduced food intake, impaired mobility, and perturbations in the production or responsiveness of catabolic and anabolic hormones, cytokines and/or proteolysis inducing factors) act in concert to contribute to muscle wasting in disease states. We cite recent evidence that insulin, glucocorticoids, thyroid hormones, and nutrients regulate the rates of ubiquitinylation of protein substrates and of proteasome-dependent proteolysis in skeletal muscle.

Role of protein intake on protein synthesis and fiber distribution in the unweighted soleus muscle.

Protein turnover in skeletal muscle is very sensitive to protein intake. To examine whether protein intake is able to affect protein synthesis in the atrophied soleus muscle, the effects of a high-protein (30%, HP) and a medium-protein (15%, MP) diet were studied in rats after 21 days of hindlimb unweighting. Three weeks of unweighting induced a sharp decrease in food intake (30%). The fractional rate of protein synthesis (ks) was determined in vivo in the slow-twitch soleus muscle by use of a flooding-dose method. With respect to pair-fed animals, a significant reduction in ks occurred (33%) in MP non-weight-bearing rats, whereas it was of lesser magnitude and not significant in HP rats. In the atrophied soleus muscle of non-weight-bearing MP rats, a large decrease (42%) in type I fiber distribution was accompanied by an increase in intermediate and type IIa fibers. By contrast, a higher percentage of type I fiber was maintained with the HP diet. However, the HP diet had no beneficial effect in preventing the decrease in either type I fiber cross-sectional area (65%) or the average decrease in absolute myofibrillar and mitochondrial volumes (69 and 52%, respectively). These results demonstrate that an HP intake did not prevent soleus muscle atrophy but may sustain protein synthesis and partly preserve fiber type distribution without affecting the ultrastructural composition of fibers. Because the circulating level of free 3,5,3'-triiodothyronine was reduced by 14% with the HP diet, this effect on fiber type distribution, and possibly protein synthesis, may involve thyroid hormones.

Assembly of the regulatory complex of the 26S proteasome.

The 19S regulatory complex (RC) of 26S proteasomes is a 900-1000 kDa particle composed of 18 distinct subunits (S1-S15) ranging in molecular mass from 25 to 110 kDa. This particle confers ATP-dependence and polyubiquitin (polyUb) recognition to the 26S proteasome. The symmetry and homogenous structure of the proteasome contrasts sharply with the remarkable complexity of the RC. Despite the fact that the primary sequences of all the subunits are now known, insight has been gained into the function of only eight subunits. The six ATPases within the RC constitute a subfamily (S4-like ATPases) within the AAA superfamily and we have shown that they form specific pairs in vitro. We have now determined that putative coiled-coils within the variable N-terminal regions of these proteins are likely to function as recognition elements that direct the proper placement of the ATPases within the RC. We have also begun mapping putative interactions between non-ATPase subunits and S4-like ATPases. These studies have allowed us to build a model for the specific arrangement of 9 subunits within the human regulatory complex. This model agrees with recent findings by Glickman et al. who have reported that two subcomplexes, termed the base and the lid, form the RC of budding yeast 26S proteasomes.

Mapping subunit contacts in the regulatory complex of the 26 S proteasome. S2 and S5b form a tetramer with ATPase subunits S4 and S7.

The 19 S regulatory complex (RC) of the 26 S proteasome is composed of at least 18 different subunits, including six ATPases that form specific pairs S4-S7, S6-S8, and S6'-S10b in vitro. One of the largest regulatory complex subunits, S2, was translated in reticulocyte lysate containing [(35)S]methionine and used to probe membranes containing SDS-polyacrylamide gel electrophoresis separated RC subunits. S2 bound to two ATPases, S4 and S7. Association of S2 with regulatory complex subunits was also assayed by co-translation and sedimentation. S2 formed an immunoprecipitable heterotrimer upon co-translation with S4 and S7. The non-ATPase S5b also formed a ternary complex with S4 and S7 and the three proteins assembled into a tetramer with S2. Neither S2 nor S5b formed complexes with S6'-S10b dimers or with S6-S8 oligomers. The use of chimeric ATPases demonstrated that S2 binds the NH(2)-terminal region of S4 and the COOH-terminal two-thirds of S7. Conversely, S5b binds the COOH-terminal two-thirds of S4 and to S7's NH(2)-terminal region. The demonstrated association of S2 with ATPases in the mammalian 19 S regulatory complex is consistent with and extends the recent finding that the yeast RC is composed of two subcomplexes, the lid and the base (Glickman, M. H., Rubin, D. M., Coux, O., Wefes, I., Pfeifer, G., Cejka, Z., Baumeister, W., Fried, V. A., and Finley, D. (1998) Cell 94, 615-623).

Adaptation of the ubiquitin-proteasome proteolytic pathway in cancer cachexia.

The ubiquitin-proteasome proteolytic pathway is of major importance in the breakdown of skeletal muscle proteins. The first step in this pathway is the covalent attachment of polyubiquitin chains to the targeted protein. Polyubiquitinylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyze recent findings in the regulation of ubiquitinylation of protein substrates and of their subsequent proteasome-dependent degradation in animal models of cancer cachexia. In particular, we discuss the influence of various mediators (anorexia, hormones, prostaglandins, cytokines, and proteolysis-inducing factor) in signaling the activation of ubiquitin-proteasome proteolysis in skeletal muscle. These findings have lead to new concepts that are starting to be used for preventing cachexia in cancer and other wasting diseases.

Regulation of proteolysis.

The mechanisms of proteolysis remain to be fully defined. This review focuses on recent advances in our understanding of the ubiquitin-proteasome-dependent pathway, which is involved in the control of many major biological functions. The ubiquitinylation/deubiquitinylation system is a complex machinery responsible for the specific tagging and proof-reading of substrates degraded by the 26S proteasome, as well as having other functions. The formation of a polyubiquitin degradation signal is required for proteasome-dependent proteolysis. Several families of enzymes, which may comprise hundreds of members to achieve high selectivity, control this process. The substrates tagged by ubiquitin are then recognized by the 26S proteasome and degraded into peptides. In addition, the 26S proteasome also recognizes and degrades some non-ubiquitinylated proteins. In fact, there are multiple ubiquitin- or proteasome-dependent pathways. These systems presumably degrade specific classes of substrates and single proteins by alternative mechanisms and could be interconnected. They may also interfere or cooperate with other proteolytic pathways.

Effects of protein supplementation during prolonged exercise at moderate altitude on performance and plasma amino acid pattern.

The effects of two levels of protein intake on muscle performance and energy metabolism were studied in humans submitted to repeated daily sessions of prolonged exercise at moderate altitude. For this purpose, 29 healthy males, were exposed to seven successive stages of ski-mountaineering at altitudes between 2500 and 3800 m, and to an isocaloric diet (4000 kcal.day-1, 16,760 kJ.day-1) with either 1.5 g.kg-1.day-1 (C group, n = 14), or 2.5 g.kg-1.day-1 (PR group, n = 15) protein intake. Measurements made after the ski-mountaineering programme did not show any change in body mass. The peak torque during maximal isometric voluntary contraction (MVC) of the quadriceps muscle was unaffected by the repeated exercises, whereas the endurance time at 50% MVC was decreased in PR subjects (-26.8%, P < 0.001). Increased levels of both free fatty acids (+ 147%, P < 0.001) and glycerol (+ 170%, P < 0.001) observed in C subjects would suggest that lipolysis was enhanced after the repeated exercise. The plasma amino acid pattern was altered after completion of the ski-mountaineering programme; the plasma concentration of the three branched-chain amino acids (BCAA) was significantly decreased in C subjects, whereas the higher level of protein intake (PR group) greatly minimized the exercise-induced decrease in serum BCAA.

A high protein diet does not improve protein synthesis in the nonweight-bearing rat tibialis anterior muscle.

We recently demonstrated that a high protein intake partially prevented the decrease in protein synthesis in the atrophied dark soleus muscle of rats that were hindlimb suspended (HS) for 21 d. To study the possible role of protein intake in a muscle more representative of the whole musculature, we measured the effect of a high protein (HP) (30%) and a medium protein (MP) (15%) diet on protein synthesis in the pale fast-twitch tibialis anterior muscle of HS rats. The HS animals were suspended by the tail for 21 d so that only their front legs were able to rest on the floor. The fractional rate of protein synthesis (Ks) was determined in vivo using a flooding dose method. A significantly lower Ks (24-25%) was found in both HS-MP and HS-HP rats compared with their pair-fed control groups. Reduced Ks in HS-MP rats relative to their pair-fed controls resulted from a decrease in the translational efficiency (KRNA, 23%, P < 0.01), while the ratio of RNA to protein (Cs) was unaffected. In contrast, the decrease in KRNA was prevented in the HS-HP animals compared with their pair-fed controls (P < 0.05). Hindlimb suspension did not alter fiber type distribution in the tibialis anterior muscle. However, a higher proportion of intermediate and Type I fibers with a concomitant decrease in Type II fibers was observed in both CT and HS animals fed the HP diet compared with those fed the MP diet (P < 0.05). These data clearly establish that depressed protein synthesis contributes to altered protein accretion in fast-twitch muscles during long-term hindlimb suspension. Although the HP diet prevented the decrease in translational efficiency in muscles from HS rats, it neither sustained protein synthesis nor prevented the reduction in muscle growth. Thus, it seems very unlikely that a high protein diet had any beneficial effect on the overall musculature during weightlessness in rats.

Regulation of ATP-ubiquitin-dependent proteolysis in muscle wasting.

Protein breakdown plays a major role in muscle growth and atrophy. However, the regulation of muscle proteolysis by nutritional, hormonal and mechanical factors remains poorly understood. In this review, the methods available to study skeletal muscle protein breakdown, and our current understanding of the role of 3 major proteolytic systems that are well characterized in this tissue (ie the lysosomal, Ca(2+)-dependent and ATP-ubiquitin-dependent proteolytic pathways) are critically analyzed. ATP-ubiquitin-dependent proteolysis is discussed in particular since recent data strongly suggest that this pathway may be responsible for the loss of myofibrillar proteins in many muscle-wasting conditions in rodents. In striking contrast to either the lysosomal or the Ca(2+)-dependent processes, ATP-ubiquitin-dependent protein breakdown is systematically influenced by nutritional manipulation (fasting and dietary protein deficiency), muscle activity and disuse (denervation atrophy and simulated weightlessness), as well as pathological conditions (sepsis, cancer, trauma and acidosis). The hormonal control of this pathway, its possible substrates, rate-limiting step, and functional associations with other proteolytic systems are discussed.

Expression of subunits of the 19S complex and of the PA28 activator in rat skeletal muscle.

A precise knowledge of the role of subunits of the 19S complex and the PA28 regulator, which associate with the 20S proteasome and regulate its peptidase activities, may contribute to design new therapeutic approaches for preventing muscle wasting in human diseases. The proteasome is mainly responsible for the muscle wasting of tumor-bearing and unweighted rats. The expression of some ATPase (MSS1, P45) and non ATPase (P112-L, P31) subunits of the 19S complex, and of the two subunits of the PA28 regulator, was studied in such atrophying muscles. The mRNA levels for all studied subunits increased in unweighted rats, and analysis of MSS1 mRNA distribution profile in polyribosomes showed that this subunit entered active translation. By contrast, only the mRNA levels for MSS1 increased in the muscles from cancer rats. Thus, gene expression of the proteasome regulatory subunits depends on a given catabolic state. Torbafylline, a xanthine derivative which inhibits tumor necrosis factor production, prevented the activation of protein breakdown and the increased expression of 20S proteasome subunits in cancer rats, without reducing the elevated MSS1 mRNA levels. Thus, the increased expression of MSS1 is regulated independently of 20S proteasome subunits, and did not result in accelerated proteolysis.

Differential regulation of the lysosomal, Ca2+-dependent and ubiquitin/proteasome-dependent proteolytic pathways in fast-twitch and slow-twitch rat muscle following hyperinsulinaemia.

In order to characterize the poorly defined mechanisms that account for the anti-proteolytic effects of insulin in skeletal muscle, we investigated in rats the effects of a 3 h systemic euglycaemic hyperinsulinaemic clamp on lysosomal, Ca(2+)-dependent proteolysis, and on ubiquitin/proteasome-dependent proteolysis. Proteolysis was measured in incubated fast-twitch mixed-fibre extensor digitorum longus (EDL) and slow-twitch red-fibre soleus muscles harvested at the end of insulin infusion. Insulin inhibited proteolysis (P<0.05) in both muscles. This anti-proteolytic effect disappeared in the presence of inhibitors of the lysosomal/Ca(2+)-dependent proteolytic pathways in the soleus, but not in the EDL, where only the proteasome inhibitor MG 132 (benzyloxycarbonyl-leucyl-leucyl-leucinal) was effective. Furthermore, insulin depressed ubiquitin mRNA levels in the mixed-fibre tibialis anterior, but not in the red-fibre diaphragm muscle, suggesting that insulin inhibits ubiquitin/proteasome-dependent proteolysis in mixed-fibre muscles only. However, depressed ubiquitin mRNA levels in such muscles were not associated with significant decreases in the amount of ubiquitin conjugates, or in mRNA levels or protein content for the 14 kDa ubiquitin-conjugating enzyme E2 and 20 S proteasome subunits. Thus alternative, as yet unidentified, mechanisms are likely to contribute to inhibit the ubiquitin/proteasome system in mixed-fibre muscles.

Manipulation of the ubiquitin-proteasome pathway in cachexia: pentoxifylline suppresses the activation of 20S and 26S proteasomes in muscles from tumor-bearing rats.

The development of pharmacological approaches for preventing the loss of muscle proteins would be extremely valuable for cachectic patients. For example, severe wasting in cancer patients correlates with a reduced efficacy of chemotherapy and radiotherapy. Pentoxifylline (PTX) is a very inexpensive xanthine derivative, which is widely used in humans as a haemorheological agent, and inhibits tumor necrosis factor transcription. We have shown here that a daily administration of PTX prevents muscle atrophy and suppresses increased protein breakdown in Yoshida sarcoma-bearing rats by inhibiting the activation of a nonlysosomal, Ca(2+)-independent proteolytic pathway. PTX blocked the ubiquitin pathway, apparently by suppressing the enhanced expression of ubiquitin, the 14-kDa ubiquitin conjugating enzyme E2, and the C2 20S proteasome subunit in muscle from cancer rats. The 19S complex and 11S regulator associate with the 20S proteasome and regulate its peptidase activities. The mRNA levels for the ATPase subunit MSS1 of the 19S complex increased in cancer cachexia, in contrast with mRNAs of other regulatory subunits. This adaptation was suppressed by PTX, suggesting that the drug inhibited the activation of the 26S proteasome. This is the first demonstration of a pharmacological manipulation of the ubiquitin-proteasome pathway in cachexia with a drug which is well tolerated in humans. Overall, the data suggest that PTX can prevent muscle wasting in situations where tumor necrosis factor production rises, including cancer, sepsis, AIDS and trauma.