Activation of ATP-ubiquitin-dependent proteolysis in skeletal muscle in vivo and murine myoblasts in vitro by a proteolysis-inducing factor (PIF)

Abstract

Loss of skeletal muscle is a major factor in the poor survival of patients with cancer cachexia. This study examines the mechanism of catabolism of skeletal muscle by a tumour product, proteolysis-inducing factor (PIF). Intravenous administration of PIF to normal mice produced a rapid decrease in body weight (1.55 ± 0.12 g in 24 h) that was accompanied by increased mRNA levels for ubiquitin, the Mr 14 000 ubiquitin carrier-protein, E2, and the C9 proteasome subunit in gastrocnemius muscle. There was also increased protein levels of the 20S proteasome core and 19S regulatory subunit, detectable by immunoblotting, suggesting activation of the ATP-ubiquitin-dependent proteolytic pathway. An increased protein catabolism was also seen in C2C12 myoblasts within 24 h of PIF addition with a bell-shaped dose–response curve and a maximal effect at 2–4 nM. The enhanced protein degradation was attenuated by anti-PIF antibody and by the proteasome inhibitors MG115 and lactacystin. Glycerol gradient analysis of proteasomes from PIF-treated cells showed an elevation in chymotrypsin-like activity, while Western analysis showed a dose-related increase in expression of MSSI, an ATPase that is a regulatory subunit of the proteasome, with a dose–response curve similar to that for protein degradation. These results confirm that PIF acts directly to stimulate the proteasome pathway in muscle cells and may play a pivotal role in protein catabolism in cancer cachexia. © 2001 Cancer Research Campaign http://www.bjcancer.com

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

“…The activity was adjusted for the protein concentration of the sample, determined using the Bradford assay (Sigma Chemical Co., Dorset, UK). Both substrate and enzyme concentrations were optimal and the results were similar to those obtained using purified proteasomes (Lorite et al, 2001). …”

“…This was measured essentially as previously described for murine myoblasts (Lorite et al, 2001). Myotubes were formed in six-well multidishes containing 2 ml DMEM and 2% HS.…”

Increased expression of phosphorylated forms of RNA-dependent protein kinase and eukaryotic initiation factor 2α may signal skeletal muscle atrophy in weight-losing cancer patients

“…Several potential mediators of the cachectic process, including proteolysis-inducing factor (PIF) and angiotensin II (Ang II), inhibit protein synthesis in skeletal muscle Russell et al, 2006a), and also stimulate degradation, through increased activity and expression of the ubiquitin -proteasome pathway (Lorite et al, 2001;Sanders et al, 2005). We have recently shown a link between the ability of PIF and Ang II to inhibit protein synthesis and increase protein degradation in murine myotubes through the dsRNA-dependent protein kinase (PKR).…”

Angiotensin II directly induces muscle protein catabolism through the ubiquitin–proteasome proteolytic pathway and may play a role in cancer cachexia

“…Further studies in rats showed that mRNA levels of the ubiquitin ligases atrogin-1 and muscle ring-finger-1 (MURF1) were upregulated in skeletal muscle by Ang II, suggesting that protein degradation was mediated through the ubiquitin -proteasome proteolytic pathway (Song et al, 2005). This raises the possibility that Ang II directly enhances expression and activity of this pathway, as does the tumour factor PIF (Lorite et al, 2001). However, the effect in vivo was considered to be indirect and mediated by intermediate factors such as glucocorticoids (Song et al, 2005).…”