NF-κB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin-proteasome system in skeletal muscle

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-kappaB (NF-kappaB) 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-kappaBalpha, an NF-kappaB inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-kappaBalpha, nuclear accumulation of NF-kappaB and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-kappaBalpha 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-kappaB. 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 beta-subunits of the proteasome, protein expression of 20S alpha-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-kappaBalpha. The ability of mutant I-kappaBalpha 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-kappaB.