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

Whitehouse AS, Khal J, Tisdale MJ - Br. J. Cancer (2003)

Bottom Line: The effect was attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA).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.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Sciences Research Institute, Aston University, Birmingham B4 7ET, UK.

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-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.

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Effect of treatment of C2C12 myotubes with 15(S)-HETE for 4 h on expression of mRNA for (A) C2 proteasome subunit, (B) C5 proteasome subunit, (C) E214k, as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m. from three separate determinations performed on different occasions with different samples. Differences are indicated as (a) P<0.05, (b) P<0.01 and (c) P<0.001 from cells treated with 0 nM 15(S)-HETE.
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fig3: Effect of treatment of C2C12 myotubes with 15(S)-HETE for 4 h on expression of mRNA for (A) C2 proteasome subunit, (B) C5 proteasome subunit, (C) E214k, as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m. from three separate determinations performed on different occasions with different samples. Differences are indicated as (a) P<0.05, (b) P<0.01 and (c) P<0.001 from cells treated with 0 nM 15(S)-HETE.

Mentions: To determine whether the increased proteasome proteolytic activity arose from an increased gene expression mRNA levels of proteasome subunits C2(α) and C5(β), as well as the ubiquitin-conjugating enzyme, E214k, were determined in C2C12 myotubes exposed to various concentrations of 15(S)-HETE for various times to determine the earliest time of gene induction and how long this persisted, using quantitative competitive RT–PCR (Figure 3Figure 3


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

Whitehouse AS, Khal J, Tisdale MJ - Br. J. Cancer (2003)

Effect of treatment of C2C12 myotubes with 15(S)-HETE for 4 h on expression of mRNA for (A) C2 proteasome subunit, (B) C5 proteasome subunit, (C) E214k, as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m. from three separate determinations performed on different occasions with different samples. Differences are indicated as (a) P<0.05, (b) P<0.01 and (c) P<0.001 from cells treated with 0 nM 15(S)-HETE.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376908&req=5

fig3: Effect of treatment of C2C12 myotubes with 15(S)-HETE for 4 h on expression of mRNA for (A) C2 proteasome subunit, (B) C5 proteasome subunit, (C) E214k, as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m. from three separate determinations performed on different occasions with different samples. Differences are indicated as (a) P<0.05, (b) P<0.01 and (c) P<0.001 from cells treated with 0 nM 15(S)-HETE.
Mentions: To determine whether the increased proteasome proteolytic activity arose from an increased gene expression mRNA levels of proteasome subunits C2(α) and C5(β), as well as the ubiquitin-conjugating enzyme, E214k, were determined in C2C12 myotubes exposed to various concentrations of 15(S)-HETE for various times to determine the earliest time of gene induction and how long this persisted, using quantitative competitive RT–PCR (Figure 3Figure 3

Bottom Line: The effect was attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA).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.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Sciences Research Institute, Aston University, Birmingham B4 7ET, UK.

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-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.

Show MeSH
Related in: MedlinePlus