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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 24 h on expression of mRNA for (A) C2 proteasome subunit, (B) C5 proteasome subunit, as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m., where n=3 representing three separate determinations on different samples. Differences from control are indicated as (b) P<0.01 and (c) P<0.001.
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fig6: Effect of treatment of C2C12 myotubes with 15(S)-HETE for 24 h on expression of mRNA for (A) C2 proteasome subunit, (B) C5 proteasome subunit, as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m., where n=3 representing three separate determinations on different samples. Differences from control are indicated as (b) P<0.01 and (c) P<0.001.

Mentions: Effect of 15(S)-HETE on the expression of mRNA for the C5 proteasome subunit in C2C12 myotubes in the absence (solid boxes) and presence (open boxes) of 50 μM EPA. Results represent mean±s.e.m. from two separate determinations performed on different occasions with different samples. Where there was no differences between individual measurements no standard error is shown. Differences from control are indicated as (b) P<0.01 and (c) P<0.001 as determined by Student's t-test.


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 24 h on expression of mRNA for (A) C2 proteasome subunit, (B) C5 proteasome subunit, as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m., where n=3 representing three separate determinations on different samples. Differences from control are indicated as (b) P<0.01 and (c) P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Effect of treatment of C2C12 myotubes with 15(S)-HETE for 24 h on expression of mRNA for (A) C2 proteasome subunit, (B) C5 proteasome subunit, as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m., where n=3 representing three separate determinations on different samples. Differences from control are indicated as (b) P<0.01 and (c) P<0.001.
Mentions: Effect of 15(S)-HETE on the expression of mRNA for the C5 proteasome subunit in C2C12 myotubes in the absence (solid boxes) and presence (open boxes) of 50 μM EPA. Results represent mean±s.e.m. from two separate determinations performed on different occasions with different samples. Where there was no differences between individual measurements no standard error is shown. Differences from control are indicated as (b) P<0.01 and (c) P<0.001 as determined by Student's t-test.

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