Signal transduction pathways involved in proteolysis-inducing factor induced proteasome expression in murine myotubes

The proteolysis-inducing factor (PIF) is produced by cachexia-inducing tumours and initiates protein catabolism in skeletal muscle. The potential signalling pathways linking the release of arachidonic acid (AA) from membrane phospholipids with increased expression of the ubiquitin-proteasome proteolytic pathway by PIF has been studied using C2C12 murine myotubes as a surrogate model of skeletal muscle. The induction of proteasome activity and protein degradation by PIF was blocked by quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor and trifluroacetyl AA, an inhibitor of cytosolic PLA2. PIF was shown to increase the expression of calcium-independent cytosolic PLA2, determined by Western blotting, at the same concentrations as those inducing maximal expression of 20S proteasome α-subunits and protein degradation. In addition, both U-73122, which inhibits agonist-induced phospholipase C (PLC) activation and D609, a specific inhibitor of phosphatidylcholine-specific PLC also inhibited PIF-induced proteasome activity. This suggests that both PLA 2 and PLC are involved in the release of AA in response to PIF, and that this is important in the induction of proteasome expression. The two tyrosine kinase inhibitors genistein and tryphostin A23 also attenuated PIF-induced proteasome expression, implicating tyrosine kinase in this process. PIF induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) at the same concentrations as that inducing proteasome expression, and the effect was blocked by PD98059, an inhibitor of MAPK kinase, as was also the induction of proteasome expression, suggesting a role for MAPK activation in PIF-induced proteasome expression. © 2003 Cancer Research UK

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 C2C12 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 μM), suggesting a requirement for new protein synthesis. In both myoblasts and myotubes protein degradation was accompanied by an increased expression of the α-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 (E214k), 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

Expression of the ubiquitin-proteasome pathway and muscle loss in experimental cancer cachexia

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E214k, has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E214k were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E214k mRNA and protein levels in gastrocnemius muscle with increases of 6–8-fold for C2 and two-fold for E214k between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25–27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18–20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E214k mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss

Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin–proteasome pathway

The potential role of 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) as an intracellular signal for increased protein catabolism and induction of the expression of key components of the ubiquitin-proteasome proteolytic pathway induced by a tumour cachectic factor, proteolysis-inducing factor has been studied in murine C2C12 myotubes. 15(S)-HETE induced protein degradation in these cells with a maximal effect at concentrations between 78 and 312 nM. The effect was attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). There was an increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome, in the same concentration range as that inducing total protein degradation, and this effect was also attenuated by EPA. 15(S)-hydroxyeicosatetraenoic acid also increased maximal expression of mRNA for proteasome subunits C2 and C5, as well as the ubiquitin-conjugating enzyme, E214k, after 4 h incubation, as determined by quantitative competitive RT-PCR. The concentrations of 15-HETE affecting gene expression were the same as those inducing protein degradation. Western blotting of cellular supernatants of myotubes treated with 15(S)-HETE for 24 h showed increased expression of p42, an ATPase subunit of the regulatory complex at similar concentrations, as well as a decrease in expression of myosin in the same concentration range. 15(S)-hydroxyeicosatetraenoic acid activated binding of nuclear factor-κB (NF-κB) in the myotube nucleus and stimulated degradation of 1-κBα. The effect on the NF-κB/1-κBα system was attenuated by EPA. In addition, the NF-κB inhibitor peptide SN50 attenuated the increased chymotrypsin-like enzyme activity in the presence of 15(S)-HETE. These results suggest that 15(S)-HETE induces degradation of myofibrillar proteins in differentiated myotubes through an induction of an increased expression of the regulatory components of the ubiquitin-proteasome proteolytic pathway possibly through the intervention of the nuclear transcription factor NF-κB, and that this process is inhibited by EPA. © 2003 Cancer Research UK

Effects of a new nitrosourea (cystemustine) on skeletal muscle protein turnover in healthy and tumor-bearing mice

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GONZÁLEZ GERALDO, J. L. (2014) Hacia una universidad más humana. ¿Es superior la educación superior? Madrid, Editorial Biblioteca Nueva.

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Selective activation of ATP-ubiquitin-dependent protein degradation in skeletal muscles proximal to the tumor of Yoshida sarcoma bearing rats

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