0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society
Vol. 82, No. 9 Printed in U.S.A.
COMMENTS Glucocorticoids Do Not Regulate the Expression of Proteolytic Genes in Skeletal Muscle from Cushing’s Syndrome Patients* ´ CILE RALLIE ` RE, IGOR TAUVERON, DANIEL TAILLANDIER, LAURENT GUY, CE JEAN-PAUL BOITEUX, BERNARD GIRAUD, DIDIER ATTAIX, AND ´ BLOT PHILIPPE THIE Institut Natı´onal de Recherche Agronomique (C.R., D.T., D.A.), Unite´ d’Etude du Me´tabolisme Azote´, and Centre de Recherche en Nutrition Humaine de Clermont-Ferrand, 63122 Ceyrat, France; Service d’Endocrinologie et Maladies Me´taboliques (I.T., P.T.), and Service d’Urologie (L.G., J.-P.B., B.G.), Centre Hospitalier Universitaire de Clermont-Ferrand, BP 69, 63003 Clermont-Ferrand Cedex, France ABSTRACT Glucocorticoids signal enhanced proteolysis in various instances of muscle atrophy and increased gene expression of components of the lysosomal, Ca21-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 Ca21-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 mechanism, preventing sustained increased protein breakdown and avoiding rapid muscle wasting. (J Clin Endocrinol Metab 82: 3161–3164, 1997)
A
pathway is now recognized as the major proteolytic process contributing to enhanced proteolysis in various instances of muscle wasting in animals (1–5, 8 –10) and in humans (11, 12). Ubiquitin covalently binds to protein substrates and marks them for degradation by the 26S proteasome complex, which contains the 20S proteasome responsible for multiple peptidase activities, and 19S regulatory complexes (13). Dexamethasone administration resulted in increased mRNA levels for critical components of the ubiquitin-proteasome pathway in rat muscle (2). Conversely, adrenalectomy suppressed the increased protein breakdown and the enhanced expression of ubiquitin in fasted rats (3). Similarly, the glucocorticoid receptor antagonist RU 38486 inhibited the sepsis-induced increase in total and myofibrillar protein breakdown rates and blunted increased ubiquitin expression (5). In vitro, this steroid-receptor antagonist also blocked the enhanced expression of ubiquitin and the C2 proteasome subunit in BC3H1 myocytes treated with dexamethasone (6). Cushing’s syndrome is an unusual but severe disease, characterized by centrally localized adipose tissue deposition, osteoporosis, hypertension, and muscle wasting. An increased urinary 3-methylhistidine excretion (an indirect index of skeletal muscle protein breakdown) was reported in Cushing’s syndrome, suggesting that enhanced proteolysis contributed to that wasting (1, 14). Because glucocorticoids up-regulated the expression of proteolytic genes in various
DMINISTRATION of glucocorticoids results in an impairment of protein synthesis and/or increased proteolysis in skeletal muscle (1, 2). Increased endogenous glucocorticoid production also activates muscle proteolysis in starvation (3) and several pathological states (4, 5). For example, glucocorticoids play a critical role in signaling increased muscle proteolysis in fasting (3) and sepsis (5). By contrast, glucocorticoids play a secondary role in mediating enhanced protein breakdown in acidosis both in vivo (4) and in vitro (6). The mechanisms by which glucocorticoids activate proteolytic systems are not yet fully understood. Glucocorticoid administration resulted in increased messenger RNA (mRNA) levels for cathepsins B and D (i.e. lysosomal proteinases), and m-calpain (a Ca21-dependent proteinase) in rat skeletal muscle (2). Furthermore, glucocorticoids up-regulated the expression of cathepsins B and D, and m-calpain, in rat L8 myotubes (7). However, the ubiquitin-proteasome Received February 25, 1997. Revision received May 29, 1997. Accepted June 2, 1997. Address all correspondence and requests for reprints to: Didier Attaix, Ph.D., INRA de Theix, Unite´ d’Etude du Me´tabolisme Azote´, 63122 Ceyrat, France. E-mail:
[email protected]. * This study was supported by grants (to D.A. and P.T.) from the French Ministe`re de l’Education Nationale, Enseignement Supe´rieur, Recherche et Insertion Professionnelle and the Institut National de la Recherche Agronomique.
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animal models of muscle atrophy (1–5), we investigated whether such an adaptation prevailed in muscle biopsies from Cushing’s syndrome patients. Subjects and Methods Patients Four patients with ACTH-independent Cushing’s syndrome and four age- and sex-matched control subjects were studied (Table 1). All patients had typical symptoms of the disease, including elevated urinary free cortisol excretion (data not shown). The dexamethasone inhibition test was negative, which supported the diagnosis. ACTH was low in all patients. An adrenal tumor was demonstrated by either computerized tomography or magnetic resonance imaging in the patients. The tumors were classified by histopathological analysis (Table 1). The body mass index of only two Cushing’s syndrome patients was high, as frequently observed in comparable studies (15, 16). The control subjects were undergoing lumbar surgical procedure for various diseases, excluding kidney failure (Table 1).
Methods Patients and control subjects underwent the same anesthesiological protocol. Biopsies of external oblique muscles were obtained during surgery between 0900 and 1000 h. They were immediately frozen in liquid nitrogen and stored at 280 C. Total RNA was extracted from approximately 100 mg of external oblique muscle, as previously described (2, 8 –11). Ten micrograms of RNA was electrophoresed in formaldehyde agarose gels (1%), transferred electrophoretically to nylon membranes (GeneScreen, NEN Research Products, Boston, MA), and covalently bound to the membrane by ultraviolet cross-linking. Membranes were hybridized with [32P]complementary DNA (cDNA) probes encoding human cathepsin D, m-calpain, and HC2 or HC8 proteasome subunits, chicken polyubiquitin, and rat 14-kDa ubiquitin conjugating enzyme E2 (14-kDa E2), as previously described (2, 8 –11). Hybridizations were conducted overnight at 65 C with [32P]cDNA fragments labeled by random priming (Oligolabelling kit, Pharmacia, Uppsala, Sweden). After washing at the same temperature, filters were autoradiographed at 280 C with intensifying screens on Hyperfilm-MP films (Amersham, UK). After stripping of the different probes, the filters were reprobed with a cDNA corresponding to mouse 18S ribosomal RNA (rRNA) (American Type Culture Collection #63178, Rockville, MD) labeled by Nick Translation (Nick Translation Kit, Boehringer Mannheim, Germany). All autoradiographic signals were quantified by digital image processing and analysis (NIH Image 1.54) on the same autoradiograms, and normalized using the corresponding 18S rRNA signals to correct for slight differences in RNA loading.
Results
The regulation of the expression of proteolytic genes in human skeletal muscle is poorly understood. We measured mRNA levels for components of the three major proteolytic pathways that are well characterized in muscle (1–5), i.e. cathepsin D (a lysosomal proteinase), m-calpain (a Ca21activated proteinase), and several proteins involved in ubiquitin-proteasome-dependent proteolysis. We recently have reported increased mRNA levels for all these proteolytic genes in skeletal muscle from head trauma patients, which correlated with negative nitrogen balance, and increased rates of whole-body and myofibrillar protein breakdown (11). These experiments strongly suggested that the ubiquitin system accounted for the breakdown of myofibrillar proteins in humans, as reported in animal studies (1, 5). An increased expression of ubiquitin and subunit HC3 of the 20S proteasome also was observed recently in skeletal muscle from septic patients (12). It was suggested that the elevated glucocorticoid production that prevails in both head trauma (11) and septic (12) patients contributed to the activation of the ubiquitin pathway. In the present study, however, we were unable to demonstrate any significant variation (P . 0.05) in mRNA levels for ubiquitin, 14-kDa E2, and subunit HC2 of the 20S proteasome in the muscles from Cushing’s syndrome patients (Fig. 1), as well as for the HC8 proteasome subunit (data not shown). These observations are in striking contrast with a glucocorticoid-induced increased expression of these critical components of the ubiquitin pathway either in muscle from intact animals (1–5) or in cultured myocytes (6). Fig. 2 shows that the expression of cathepsin D and m-calpain was also unaffected in the muscle biopsies from Cushing’s syndrome patients (P . 0.05). This is again in contrast with previous in
Statistical analysis Data are expressed as means 6 sem. The significance of differences was analyzed by Student’s t test.
TABLE 1. Characteristics of patients Age (yr)
Cushing’s syndrome 54 45 40 63 Control patients 60 44 38 30
Sex
Body mass index (kg/m2)
Diagnosis
patients F F F M
23.6 24.2 29.7 30.0
adrenal adrenal adrenal adrenal
F F F M
25.4 22.2 23.2 23.0
hematoma lithiasis hydronephrosis hydronephrosis
F, Female; M, male.
adenoma adenoma carcinoma adenoma FIG. 1. Quantification of mRNA levels for ubiquitin, 14-kDa E2, and proteasomal subunit HC2 in external oblique muscle from control (black bars) and Cushing’s syndrome patients (hatched bars). Autoradiographic signals were corrected for 18S rRNA abundance. Data are means 6 SEM (vertical bars) for four patients. Representative Northern blots also are shown.
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components of the ubiquitin pathway in muscle biopsies from Duchenne muscular dystrophy patients (19). Thus, it seems that in chronic situations of human muscle wasting, such as dystrophies or Cushing’s syndrome, there is no sustained adaptation in gene expression of components of the ubiquitin-proteasome pathway, in contrast with acute catabolic states (11, 12). Finally, an acute infusion of cortisol results in increased whole-body proteolysis in humans (20), and there are some indirect arguments suggesting an increased rate of muscle proteolysis in Cushing’s syndrome patients (14). However, no change (15) or even a decrease (21) in whole-body protein breakdown rates of Cushing’s syndrome patients was also reported, suggesting that muscle proteolysis was either unchanged or decreased. These observations would be more consistent with our data, because the expression of proteolytic genes was unchanged or tended to decrease, although not significantly [see the mRNA levels for ubiquitin and 14-kDa E2 (Fig. 1) or cathepsin D (Fig. 2)], in the muscle biopsies from our patients. Acknowledgments FIG. 2. Quantification of mRNA levels for cathepsin D and m-calpain in external oblique muscle from control (black bars) and Cushing’s syndrome patients (hatched bars). Autoradiographic signals were corrected for 18S rRNA abundance. Data are means 6 SEM (vertical bars) for four patients. Representative Northern blots also are shown.
vivo (2) and in vitro studies (7). It can be argued that we measured the expression of proteolytic genes in only a limited number of patients. However, variability was small. For example, the mean coefficient of variation for ubiquitin and the HC2 proteasome subunit mRNA levels was 24 and 29%, respectively. Such coefficients of variation were similar or smaller than in previous experiments performed with larger groups of subjects (78 and 29% in five head trauma patients (11), and approximately 40 and 50% in seven septic patients (12) for ubiquitin and a proteasome subunit, respectively). Discussion
The lack of increased mRNA levels for m-calpain in the Cushing’s syndrome patients is surprising because a glucocorticoid response element has been detected upstream from the human m-calpain promoter (7). Such a response element also was described upstream from the chicken polyubiquitin UbII gene (17), but it is unknown whether similar findings prevail in humans. Assuming that the expression of human proteolytic genes reflects either proteolytic activities (8, 10) or rates of protein breakdown (1–5, 8 –10), as reported in animal studies, the lack of detectable activation of the different degradative pathways in the muscles from Cushing’s syndrome patients is consistent with several hypotheses. First, this could be related to very slow changes in muscle mass that occur in such conditions. Second, some adaptative regulatory mechanisms may prevent sustained increased protein breakdown to avoid continuous rapid muscle wasting. Indeed, prolonged corticosterone administration to rats only resulted in a transient stimulation of muscle protein breakdown (18). We also were unable to detect any change in mRNA levels for
We would like to thank Dr. Keiji Tanaka (The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) for the gift of the plasmids encoding the human proteasome subunits, Dr. Simon S. Wing (McGill University, Montre´al, Canada) for the rat 14-kDa E2 cDNA, and Dr. Susan E. Samuels for helpful discussions.
References 1. Mitch WE, Goldberg AL. 1996 Mechanisms of muscle wasting. The role of the ubiquitin-proteasome pathway. N Engl J Med. 335:1897–1905. 2. Dardevet D, Sornet C, Taillandier D, Savary I, Attaix D, Grizard J. 1995 Sensitivity and protein turnover response to glucocorticoids are different in skeletal muscle from adult and old rats. Lack of regulation of the ubiquitinproteasome proteolytic pathway in aging. J Clin Invest. 96:2113–2119. 3. Wing SS, Goldberg AL. 1993 Glucocorticoids activate the ATP-ubiquitindependent proteolytic system in skeletal muscle during fasting. Am J Physiol. 264:E668 –E676. 4. Price SR, England BK, Bailey JL, Van Vreede K, Mitch WE. 1994 Acidosis and glucocorticoids concomitantly increase ubiquitin and proteasome subunit mRNA levels in rat muscles. Am J Physiol. 267:C955–C960. 5. Tiao G, Fagan J, Roegner V, et al. 1996 Energy-ubiquitin-dependent muscle proteolysis during sepsis is regulated by glucocorticoids. J Clin Invest. 97:339 –348. 6. Isozaki Y, Mitch WE, England BK, Price SR. 1996 Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification. Proc Natl Acad Sci USA. 93:1967–1971. 7. Hong D-H, Forsberg NE. 1995 Effects of dexamethasone on protein degradation and protease gene expression in rat L8 myotube cultures. Mol Cell Endocrinol. 108:199 –209. 8. Temparis S, Asensi M, Taillandier D, et al. 1994 Increased ATP-ubiquitindependent proteolysis in skeletal muscles of tumor-bearing rats. Cancer Res. 54:5568 –5573. 9. Voisin L, Breuille´ D, Combaret L, et al. 1996 Muscle wasting in a rat model of long lasting sepsis results from the activation of lysosomal, Ca21-activated and ubiquitin-proteasome proteolytic pathways. J Clin Invest. 97:1610 –1617. 10. Taillandier D, Aurousseau E, Meynial-Denis D, et al. 1996 Coordinate activation of lysosomal, Ca21-activated and ATP-ubiquitin-dependent proteinases in the unweighted rat soleus muscle. Biochem J. 316:65–72. 11. Mansoor O, Beaufre`re B, Boirie Y, et al. 1996 Increased mRNA levels for components of the lysosomal, Ca 21-activated and ATP-ubiquitin-dependent proteolytic pathways in skeletal muscle from head trauma patients. Proc Natl Acad Sci USA. 93:2714 –2718. 12. Tiao G, Hobler S, Wang JJ, et al. 1997 Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle. J Clin Invest. 99:163–168. 13. Coux O, Tanaka K, Goldberg AL. 1996 Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem. 65:801– 847. 14. Khaleeli AA, Edwards RHT, Gohil K, et al. 1983 Corticosteroid myopathy: a clinical and pathological study. Clin Endocrinol. 18:155–166. 15. Tessari P, Inchiostro S, Biolo G, et al. 1989 Leucine kinetics and the effects of
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hyperinsulinemia in patients with Cushing’s syndrome. J Clin Endocrinol Metab. 68:256 –262. 16. Bowes SB, Umpleby M, Cummings MH, et al. 1997 The effect of recombinant human growth hormone on glucose and leucine metabolism in Cushing’s syndrome. J Clin Endocrinol Metab. 82:243–246. 17. Mezquita J, Lopez-Ibor B, Pau M, Mezquita C. 1993 Intron and intronless transcription of the chicken polyubiquitin gene. FEBS Lett. 319:244 –248. 18. Bowes SB, Jackson NC, Papachristodoulou D, Umpleby AM, So¨nksen PH. 1996 Effect of corticosterone on protein degradation in isolated rat soleus and extensor digitorum longus muscles. J Endocrinol. 148:501–507.
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19. Combaret L, Taillandier D, Voisin L, Samuels SE, Boespflug-Tanguy O, Attaix D. 1996 No alteration in gene expression of components of the ubiquitinproteasome proteolytic pathway in dystrophin-deficient muscles. FEBS Lett. 393:292–296. 20. Simmons PS, Miles JM, Gerich JE, Haymond MW. 1984 Increased proteolysis. An effect of increases in plasma cortisol within the physiologic range. J Clin Invest. 73:412– 420. 21. Bowes SB, Benn JJ, Scobie IN, Umpleby AM, Lowy C, So¨nksen PH. 1993 Leucine metabolism in patients with Cushing’s syndrome before and after successful treatment. Clin Endocrinol. 39:591–598.
