NF-kappaB 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 C(2)C(12) 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.
Wyke
S M
SM
Pharmaceutical Sciences Research Institute, Aston University, Birmingham B4 7ET, UK.
Tisdale
M J
MJ
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D015153:Blotting, Western; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D005822:Genetic Vectors; D020672:I-kappa B Proteins / Q000378:metabolism*; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D032446:Myoblasts / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011509:Proteoglycans; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D015854:Up-Regulation
D001798:Blood Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D011509:Proteoglycans; D025801:Ubiquitin; C042057:proteolysis-inducing peptide; D000072000:NF-KappaB Inhibitor alpha; D008156:Luciferases; D046988:Proteasome Endopeptidase Complex