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

Abstract

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-kB (NFkB) 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-kBa, an NF-kB inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-kBa, nuclear accumulation of NF-kB and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-kBa 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-kB. Proteolysis-inducing factor also induced increased expression of the ubiquitinproteasome pathway, as determined by 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the b-subunits of the proteasome, protein expression of 20S a-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-kBa. The ability of mutant I-kBa 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-kB.

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

“…Phosphorylation of eIF2a has also been shown to be responsible for the inhibition of protein synthesis in rat liver by vasopressin (Kimball and Jefferson, 1990), and rat skeletal muscle by interleukin-1 (Cooney et al, 1999). Phosphorylation of PKR would also be expected to lead to an increased breakdown of myofibrillar proteins in skeletal muscle by induction of the ubiquitin -proteasome pathway (Wyke and Tisdale, 2005;Russell et al, 2006b) through activation of NF-kB (Zamanian-Daryoush et al, 2000) analogous to the effect of PIF and Ang II .…”

“…Mutation of PKR also completely attenuated the induction of protein degradation and upregulation of the ubiquitinproteasome pathway. Induction of the ubiquitin -proteasome pathway by both PIF (Wyke and Tisdale, 2005) and Ang II (Russell et al, 2006b) requires activation of the transcription factor nuclear factor-kB (NF-kB). dsRNA-dependent protein kinase has been shown to activate the upstream kinase IkB kinase (IKK) leading to degradation of the inhibitor protein IkB, leading to release of NF-kB, which migrates to the nucleus and induces transcriptional activation of specific genes (Zamanian-Daryoush et al, 2000).…”

Skeletal Muscle Atrophy, a Link between Depression of Protein Synthesis and Increase in Degradation

“…Induction of the ubiquitin-proteasome pathway in response to both PIF (9) and Ang II (8) requires activation of the nuclear transcription factor NF-B. Release of NF-B from the inactive complex with I-B in the cytosol requires phosphorylation of I-B, leading to degradation of the I-B and movement of the free NF-B into the nucleus.…”

“…Electrophoretic mobility shift assay (EMSA) kits were from Panomics (Redwood City, CA). The method for the transplantation of the MAC16 tumor has been described (9). All animal experiments were approved by the British Home Office, and the ethical guidelines that were followed meet the standards required by the United Kingdom Coordinating Committee on Cancer Research guidelines.…”

“…Both agents stimulate protein degradation through an increased expression and activity of the ubiquitin-proteasome proteolytic pathway (7,8), by a similar mechanism, involving activation of the nuclear transcription factor NF-B (8,9). For both PIF (7) and Ang II (8) protein degradation is inhibited by specific proteasome inhibitors, while mutations in IB␣, which prevent activation of NF-B and the ubiquitin proteasome pathway (9), also inhibit protein degradation by PIF, confirming a causal relationship. Much less is known about how these agents affect protein synthesis, although with PIF, at least, this has been suggested to arise from an effect on translation efficiency (4).…”