Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin-proteasome pathway.
The potential role of 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) as an intracellular signal for increased protein catabolism and induction of the expression of key components of the ubiquitin-proteasome proteolytic pathway induced by a tumour cachectic factor, proteolysis-inducing factor has been studied in murine C(2)C(12) myotubes. 15(S)-HETE induced protein degradation in these cells with a maximal effect at concentrations between 78 and 312 nM. The effect was attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). There was an increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome, in the same concentration range as that inducing total protein degradation, and this effect was also attenuated by EPA. 15(S)-hydroxyeicosatetraenoic acid also increased maximal expression of mRNA for proteasome subunits C2 and C5, as well as the ubiquitin-conjugating enzyme, E2(14k), after 4 h incubation, as determined by quantitative competitive RT-PCR. The concentrations of 15-HETE affecting gene expression were the same as those inducing protein degradation. Western blotting of cellular supernatants of myotubes treated with 15(S)-HETE for 24 h showed increased expression of p42, an ATPase subunit of the regulatory complex at similar concentrations, as well as a decrease in expression of myosin in the same concentration range. 15(S)-hydroxyeicosatetraenoic acid activated binding of nuclear factor-kappaB (NF-kappaB) in the myotube nucleus and stimulated degradation of I-kappaBalpha. The effect on the NF-kappaB/I-kappaBalpha system was attenuated by EPA. In addition, the NF-kappaB inhibitor peptide SN50 attenuated the increased chymotrypsin-like enzyme activity in the presence of 15(S)-HETE. These results suggest that 15(S)-HETE induces degradation of myofibrillar proteins in differentiated myotubes through an induction of an increased expression of the regulatory components of the ubiquitin-proteasome proteolytic pathway possibly through the intervention of the nuclear transcription factor NF-kappaB, and that this process is inhibited by EPA.
Whitehouse
A S
AS
Pharmaceutical Sciences Research Institute, Aston University, Birmingham B4 7ET, UK.
Khal
J
J
Tisdale
M J
MJ
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D015118:Eicosapentaenoic Acid / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014452:Ubiquitins / Q000378:metabolism*
D017931:DNA Primers; D006893:Hydroxyeicosatetraenoic Acids; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; D014452:Ubiquitins; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D015118:Eicosapentaenoic Acid; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D009218:Myosins