Development of an in-vitro model system to investigate the mechanism of muscle protein catabolism induced by proteolysis-inducing factor.
The mechanism of muscle protein catabolism induced by proteolysis-inducing factor, produced by cachexia-inducing murine and human tumours has been studied in vitro using C(2)C(12) myoblasts and myotubes. In both myoblasts and myotubes protein degradation was enhanced by proteolysis-inducing factor after 24 h incubation. In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor. Protein degradation induced by proteolysis-inducing factor was completely attenuated in the presence of cycloheximide (1 microM), suggesting a requirement for new protein synthesis. In both myoblasts and myotubes protein degradation was accompanied by an increased expression of the alpha-type subunits of the 20S proteasome as well as functional activity of the proteasome, as determined by the 'chymotrypsin-like' enzyme activity. There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E2(14k)), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor. These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia.
Gomes-Marcondes
M C C
MC
Department of Physiology and Biophysics, University of Campinas, UNICAMP, SP, Brazil 13083-970. M.J.Tisdale@aston.ac.uk
Smith
H J
HJ
Cooper
J C
JC
Tisdale
M J
MJ
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001798:Blood Proteins / Q000494:pharmacology*; D002100:Cachexia / Q000378:metabolism; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation; D046911:Macromolecular Substances; D051379:Mice; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D011509:Proteoglycans; D025801:Ubiquitin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009124:Muscle Proteins; D021122:Protein Subunits; D011509:Proteoglycans; D025801:Ubiquitin; C042057:proteolysis-inducing peptide; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C119011:26S proteasome non-ATPase regulatory subunit 13; D000251:Adenosine Triphosphatases; D018995:Myosin Heavy Chains