Induction of protein degradation in skeletal muscle by a phorbol ester involves upregulation of the ubiquitin-proteasome proteolytic pathway.
Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose-response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 microM), suggesting that it was mediated through the ubiquitin-proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin-proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome alpha-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E2(14k), also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin-proteasome pathway. TPA also induced degradation of the inhibitory protein, I-kappaBalpha, and increased nuclear accumulation of nuclear factor-kappaB (NF-kappaB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-kappaB activation by resveratrol (30 microM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-kappaB.
Wyke
S M
SM
Biomedicinal Chemistry Research Group, School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK.
Tisdale
M J
MJ
D000818:Animals; D002478:Cells, Cultured; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D032448:Myoblasts, Skeletal / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000633:toxicity*; D025801:Ubiquitin / Q000378:metabolism*
D016328:NF-kappa B; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D013755:Tetradecanoylphorbol Acetate