| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Comments |
Institut Natíonal de Recherche Agronomique (C.R., D.T., D.A.), Unité d’Etude du Métabolisme Azoté, and Centre de Recherche en Nutrition Humaine de Clermont-Ferrand, 63122 Ceyrat, France; Service d’Endocrinologie et Maladies Métaboliques (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
Address all correspondence and requests for reprints to: Didier Attaix, Ph.D., INRA de Theix, Unité d’Etude du Métabolisme Azoté, 63122 Ceyrat, France. E-mail: attaix{at}clermont.inra.fr
Abstract
Glucocorticoids signal enhanced proteolysis in various instances of muscle atrophy and increased gene expression of components of the lysosomal, Ca2+-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 Ca2+-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.
ADMINISTRATION 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 Ca2+-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 pathway is now recognized as the major proteolytic process contributing to enhanced proteolysis in various instances of muscle wasting in animals (1, 2, 3, 4, 5, 8, 9, 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 animal models of muscle atrophy (1, 2, 3, 4, 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
).
|
). 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 -80 C.
Total RNA was extracted from approximately 100 mg of external oblique muscle, as previously described (2, 8, 9, 10, 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, 9, 10, 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 -80 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.
Statistical analysis
Data are expressed as means ± SEM. The significance of differences was analyzed by Student’s t test.
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, 2, 3, 4, 5), i.e. cathepsin D (a lysosomal proteinase), m-calpain (a Ca2+-activated 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, 2, 3, 4, 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 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).
|
|
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, 2, 3, 4, 5, 8, 9, 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 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
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, Montréal, Canada) for the rat 14-kDa E2 cDNA, and Dr. Susan E. Samuels for helpful discussions.
Footnotes
1 This study was supported by grants (to D.A. and P.T.) from the
French Ministère de l’Education Nationale, Enseignement
Supérieur, Recherche et Insertion Professionnelle and the
Institut National de la Recherche Agronomique. 
Received February 25, 1997.
Revised May 29, 1997.
Accepted June 2, 1997.
References
This article has been cited by other articles:
 |
|
 |
|
  R.T. Jagoe and M.P.K.J. Engelen Muscle wasting and changes in muscle protein metabolism in chronic obstructive pulmonary disease Eur. Respir. J., November 2, 2003; 22(46_suppl): 52s - 63s. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Pirlich, H. Biering, H. Gerl, M. Ventz, B. Schmidt, S. Ertl, and H. Lochs Loss of Body Cell Mass in Cushing's Syndrome: Effect of Treatment J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1078 - 1084. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
