¹Pharmaceutical Sciences Research Institute, Aston University, Birmingham, B4 7ET, UK

Correspondence: Professor MJ Tisdale, E-mail: m.j.tisdale@aston.ac.uk

Revised 9 December 2004; Accepted 17 December 2004; Published online 15 February 2005.

Abstract

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-B (NF-B) in regulating muscle protein degradation and expression of the ubiquitin–proteasome proteolytic pathway in response to a tumour cachectic factor, proteolysis-inducing factor (PIF), has been studied by creating stable, transdominant-negative, muscle cell lines. Murine C₂C₁₂ myoblasts were transfected with plasmids with a CMV promoter that had mutations at the serine phosphorylation sites required for degradation of I-B, an NF-B inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-B, nuclear accumulation of NF-B and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-B proteins. Proteolysis-inducing factor also induced total protein degradation and loss of the myofibrillar protein myosin in myotubes containing wild-type, but not mutant, plasmids at the same concentrations as those causing activation of NF-B. Proteolysis-inducing factor also induced increased expression of the ubiquitin–proteasome pathway, as determined by 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the -subunits of the proteasome, protein expression of 20S -subunits and the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme, E2_14k, in cells containing wild-type, but not mutant, I-B. The ability of mutant I-B to inhibit PIF-induced protein degradation, as well as expression of the ubiquitin–proteasome pathway, confirms that both of these responses depend on initiation of transcription by NF-B.