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Increased expression of the ubiquitin-proteasome pathway in murine myotubes by proteolysis-inducing factor (PIF) is associated with activation of the transcription factor NF-kappaB.

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

Bottom Line: Higher concentrations of PIF had no effect.At a concentration of 4 nM, PIF induced a transient decrease in IkappaBalpha levels after 30 min incubation, while no effect was seen at 20 nM PIF.The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-kappaB accumulation in the nucleus.

View Article: PubMed Central - PubMed

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

ABSTRACT
Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C(2)C(12) myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 20S proteasome alpha-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 microM). At a concentration of 4 nM, PIF induced a transient decrease in IkappaBalpha levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IkappaBalpha, an NF-kappaB inhibitory protein, returned to normal after 60 min. Depletion of IkappaBalpha from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-kappaB/IkappaB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-kappaB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-kappaB inhibitor peptide SN50, suggesting that NF-kappaB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-kappaB accumulation in the nucleus.

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(A) Effect of PIF on the electrophoretic mobility of (γ32P) NFκB in C2C12 myotubes in the absence (lanes 1–5) and presence (lanes 6–10) of 50 μM EPA. The following additions were made: lanes 1 and 60 nM PIF; lanes 2 and 7, 4 nM PIF; lanes 3 and 8, 8 nM PIF, lanes 4 and 9, 16 nM PIF and lanes 5 and 10, 20 nM PIF. Lane 11 contains a competitor, that is an equal concentration of unlabelled NF-κB, while lane 12 contains a noncompetitor, an equal concentration of an unlabelled, unrelated oligonucleotide, usually AP2. (B) Densitometric analysis of the blot shown in (A) Values shown are in the absence (black boxes) or presence (hatched boxes) of 50 μM EPA. The blot shown is representative of three separate blots performed on different occasions. (C) The effect of SN50 on PIF-induced upregulation of the chymotrypsin-like activity of the proteasome measured after 24 h incubation. Solid boxes indicate PIF alone, hatched boxes indicate PIF+18 μM SN50. The SN50 was added 2 h prior to PIF and remained in the culture medium throughout the experiment. Sample size per treatment group n=9 and the experiment was repeated twice. Differences from control and SN50 treated cells are shown as c, P<0.001.
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fig5: (A) Effect of PIF on the electrophoretic mobility of (γ32P) NFκB in C2C12 myotubes in the absence (lanes 1–5) and presence (lanes 6–10) of 50 μM EPA. The following additions were made: lanes 1 and 60 nM PIF; lanes 2 and 7, 4 nM PIF; lanes 3 and 8, 8 nM PIF, lanes 4 and 9, 16 nM PIF and lanes 5 and 10, 20 nM PIF. Lane 11 contains a competitor, that is an equal concentration of unlabelled NF-κB, while lane 12 contains a noncompetitor, an equal concentration of an unlabelled, unrelated oligonucleotide, usually AP2. (B) Densitometric analysis of the blot shown in (A) Values shown are in the absence (black boxes) or presence (hatched boxes) of 50 μM EPA. The blot shown is representative of three separate blots performed on different occasions. (C) The effect of SN50 on PIF-induced upregulation of the chymotrypsin-like activity of the proteasome measured after 24 h incubation. Solid boxes indicate PIF alone, hatched boxes indicate PIF+18 μM SN50. The SN50 was added 2 h prior to PIF and remained in the culture medium throughout the experiment. Sample size per treatment group n=9 and the experiment was repeated twice. Differences from control and SN50 treated cells are shown as c, P<0.001.

Mentions: C2C12 myotubes were treated with EPA and PIF as described in Figure 5AFigure 5


Increased expression of the ubiquitin-proteasome pathway in murine myotubes by proteolysis-inducing factor (PIF) is associated with activation of the transcription factor NF-kappaB.

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

(A) Effect of PIF on the electrophoretic mobility of (γ32P) NFκB in C2C12 myotubes in the absence (lanes 1–5) and presence (lanes 6–10) of 50 μM EPA. The following additions were made: lanes 1 and 60 nM PIF; lanes 2 and 7, 4 nM PIF; lanes 3 and 8, 8 nM PIF, lanes 4 and 9, 16 nM PIF and lanes 5 and 10, 20 nM PIF. Lane 11 contains a competitor, that is an equal concentration of unlabelled NF-κB, while lane 12 contains a noncompetitor, an equal concentration of an unlabelled, unrelated oligonucleotide, usually AP2. (B) Densitometric analysis of the blot shown in (A) Values shown are in the absence (black boxes) or presence (hatched boxes) of 50 μM EPA. The blot shown is representative of three separate blots performed on different occasions. (C) The effect of SN50 on PIF-induced upregulation of the chymotrypsin-like activity of the proteasome measured after 24 h incubation. Solid boxes indicate PIF alone, hatched boxes indicate PIF+18 μM SN50. The SN50 was added 2 h prior to PIF and remained in the culture medium throughout the experiment. Sample size per treatment group n=9 and the experiment was repeated twice. Differences from control and SN50 treated cells are shown as c, P<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376944&req=5

fig5: (A) Effect of PIF on the electrophoretic mobility of (γ32P) NFκB in C2C12 myotubes in the absence (lanes 1–5) and presence (lanes 6–10) of 50 μM EPA. The following additions were made: lanes 1 and 60 nM PIF; lanes 2 and 7, 4 nM PIF; lanes 3 and 8, 8 nM PIF, lanes 4 and 9, 16 nM PIF and lanes 5 and 10, 20 nM PIF. Lane 11 contains a competitor, that is an equal concentration of unlabelled NF-κB, while lane 12 contains a noncompetitor, an equal concentration of an unlabelled, unrelated oligonucleotide, usually AP2. (B) Densitometric analysis of the blot shown in (A) Values shown are in the absence (black boxes) or presence (hatched boxes) of 50 μM EPA. The blot shown is representative of three separate blots performed on different occasions. (C) The effect of SN50 on PIF-induced upregulation of the chymotrypsin-like activity of the proteasome measured after 24 h incubation. Solid boxes indicate PIF alone, hatched boxes indicate PIF+18 μM SN50. The SN50 was added 2 h prior to PIF and remained in the culture medium throughout the experiment. Sample size per treatment group n=9 and the experiment was repeated twice. Differences from control and SN50 treated cells are shown as c, P<0.001.
Mentions: C2C12 myotubes were treated with EPA and PIF as described in Figure 5AFigure 5

Bottom Line: Higher concentrations of PIF had no effect.At a concentration of 4 nM, PIF induced a transient decrease in IkappaBalpha levels after 30 min incubation, while no effect was seen at 20 nM PIF.The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-kappaB accumulation in the nucleus.

View Article: PubMed Central - PubMed

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

ABSTRACT
Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C(2)C(12) myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 20S proteasome alpha-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 microM). At a concentration of 4 nM, PIF induced a transient decrease in IkappaBalpha levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IkappaBalpha, an NF-kappaB inhibitory protein, returned to normal after 60 min. Depletion of IkappaBalpha from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-kappaB/IkappaB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-kappaB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-kappaB inhibitor peptide SN50, suggesting that NF-kappaB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-kappaB accumulation in the nucleus.

Show MeSH
Related in: MedlinePlus