Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin–proteasome pathway

Abstract

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-kB (NF-kB) in the myotube nucleus and stimulated degradation of I-kBa. The effect on the NF-kB/I-kBa system was attenuated by EPA. In addition, the NF-kB inhibitor peptide SN50 attenuated the increased chymotrypsinlike 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 ubiquitinproteasome proteolytic pathway possibly through the intervention of the nuclear transcription factor NF-kB, and that this process is inhibited by EPA.

Toward a Core Nutraceutical Program for Cancer Management

“…Preincubation with EPA inhibits the impact of PIF both proximally and distally to the production of 15-HETE; not unlikely, EPA can act as a functional antagonist of arachidonic acid in 15-HETE synthesis. 127,130 However, EPA also inhibits the proteolysis triggered by addition of 15-HETE to these myotubes. The anticatabolic agent β-hydroxy-β-methylbutyrate (HMB; a leucine metabolite found to have a poorly understood favorable impact on the muscle mass and strength of individuals engaged in exercise training [131][132][133] ) also suppressed proteolysis in this model; its impact was distal to 15-HETE but proximal to PKC activation.…”

Mechanism of the Attenuation of Proteolysis-Inducing Factor Stimulated Protein Degradation in Muscle by β-Hydroxy-β-Methylbutyrate

“…Low concentrations (25 mol/L) of HMB combined with higher concentrations of PIF (10 nmol/L) appear to increase release of AA, which may account for the stimulation of protein breakdown and expression of the ubiquitin-proteasome pathway at these concentrations. PIF induced protein degradation with a parabolic doseresponse curve as reported previously (10,12,13). The shape of the dose-response curve is thought to result from the requirement of PKC in the signaling pathway (14).…”

“…EPA (99%) as free acid was purchased from Sigma-Aldridge (St. Louis, MO). EPA was used at a concentration of 50 mol/L, which previously has been shown to be effective in attenuating the action of PIF (10,12,13). HMB (as the calcium salt) was obtained from Organic Technologies Inc. (Coshocton, OH).…”

“…The full details of this pathway still have to be elucidated, but the first step involves the release of arachidonic acid from membrane phospholipids and the rapid metabolism to a range of eicosanoids, of which 15-hydroxyeicosatetraenoic acid (15-HETE) alone is capable of inducing protein degradation (10), by increasing proteasome expression (12). EPA attenuates the release of arachidonic acid and its metabolism to 15-HETE (10).…”

Potential exercise countermeasures to attenuate skeletal muscle deterioration in space

“…14 Increased activity and expression of the Ubpathway appears to be a common finding in conditions which elicit increased muscular proteolysis 15,16 including simulated antigravity models 17 and a general lowering of physical activity 14 . The similarity of these conditions indicates that the Ub-pathway may be activated during space flight as well.…”