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

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 sk eletal muscle by a tumour product, proteolysis-inducing factor (PIF). Intra venous administration of PIF to normal mice produced a rapid decrease in bo dy 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 pro tein levels of the 20S proteasome core and 19S regulatory subunit, detectab le 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 cur ve and a maximal effect at 2-4 nM. The enhanced protein degradation was att enuated by anti-PIF antibody and by the proteasome inhibitors MG115 and lac tacystin, Glycerol gradient analysis of proteasomes from PIF-treated cells showed an elevation in chymotrypsin-like activity, while Western analysis s howed a dose-related increase in expression of MSSI, an ATPase that is a re gulatory subunit of the proteasome, with a dose-response curve similar to t hat for protein degradation. These results confirm that PIF acts directly t o stimulate the proteasome pathway in muscle cells and may play a pivotal r ole in protein catabolism in cancer cachexia. (C) 2001 Cancer Research Camp aign.