Limits...
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.

Show MeSH

Related in: MedlinePlus

Western blot analysis of 20S proteasome α-subunit expression (A) or (C) the 19S subunit, p42 in C2C12 myotubes 24 h after addition of PIF alone at 0 nM (lane 1), 2 nM (lane 2), 4 nM (lane 3), 8 nM (lane 4), 16 nM (lane 5) or 40 nM (lane 6) or after pretreatment with 50 μM EPA for 2 h and subsequent treatment with PIF at 0 nM (lane 7), 2 nM (lane 8), 4 nM (lane 9), 8 nM (lane 10), 16 nM (lane 11) and 40 nM (lane 12). (B) Densitometric analysis of the blot shown in (A), n=2. (D) Densitometric analysis of the blot shown in (C), n=2. Values shown are in the absence (black boxes) or presence (stippled boxes) of 50 μM EPA. Differences from control are shown as a, P<0.001, while differences from PIF-treated cells are shown as b, P<0.001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2376944&req=5

fig3: Western blot analysis of 20S proteasome α-subunit expression (A) or (C) the 19S subunit, p42 in C2C12 myotubes 24 h after addition of PIF alone at 0 nM (lane 1), 2 nM (lane 2), 4 nM (lane 3), 8 nM (lane 4), 16 nM (lane 5) or 40 nM (lane 6) or after pretreatment with 50 μM EPA for 2 h and subsequent treatment with PIF at 0 nM (lane 7), 2 nM (lane 8), 4 nM (lane 9), 8 nM (lane 10), 16 nM (lane 11) and 40 nM (lane 12). (B) Densitometric analysis of the blot shown in (A), n=2. (D) Densitometric analysis of the blot shown in (C), n=2. Values shown are in the absence (black boxes) or presence (stippled boxes) of 50 μM EPA. Differences from control are shown as a, P<0.001, while differences from PIF-treated cells are shown as b, P<0.001.

Mentions: Effect of PIF on the chymotrypsin-like enzyme activity in C2C12 myotubes in the absence (black boxes) or presence (grey boxes) of 50 μM EPA (grey boxes). The data are expressed as mean±s.e.m. where n=9 and the values represent the activity inhibited by 10 μM lactacystin. The differences from control are indicated as a, P<0.05 and b, P<0.001, while differences in the presence of EPA are indicated as c, P<0.05 and d, P<0.001.


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)

Western blot analysis of 20S proteasome α-subunit expression (A) or (C) the 19S subunit, p42 in C2C12 myotubes 24 h after addition of PIF alone at 0 nM (lane 1), 2 nM (lane 2), 4 nM (lane 3), 8 nM (lane 4), 16 nM (lane 5) or 40 nM (lane 6) or after pretreatment with 50 μM EPA for 2 h and subsequent treatment with PIF at 0 nM (lane 7), 2 nM (lane 8), 4 nM (lane 9), 8 nM (lane 10), 16 nM (lane 11) and 40 nM (lane 12). (B) Densitometric analysis of the blot shown in (A), n=2. (D) Densitometric analysis of the blot shown in (C), n=2. Values shown are in the absence (black boxes) or presence (stippled boxes) of 50 μM EPA. Differences from control are shown as a, P<0.001, while differences from PIF-treated cells are shown as b, P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Western blot analysis of 20S proteasome α-subunit expression (A) or (C) the 19S subunit, p42 in C2C12 myotubes 24 h after addition of PIF alone at 0 nM (lane 1), 2 nM (lane 2), 4 nM (lane 3), 8 nM (lane 4), 16 nM (lane 5) or 40 nM (lane 6) or after pretreatment with 50 μM EPA for 2 h and subsequent treatment with PIF at 0 nM (lane 7), 2 nM (lane 8), 4 nM (lane 9), 8 nM (lane 10), 16 nM (lane 11) and 40 nM (lane 12). (B) Densitometric analysis of the blot shown in (A), n=2. (D) Densitometric analysis of the blot shown in (C), n=2. Values shown are in the absence (black boxes) or presence (stippled boxes) of 50 μM EPA. Differences from control are shown as a, P<0.001, while differences from PIF-treated cells are shown as b, P<0.001.
Mentions: Effect of PIF on the chymotrypsin-like enzyme activity in C2C12 myotubes in the absence (black boxes) or presence (grey boxes) of 50 μM EPA (grey boxes). The data are expressed as mean±s.e.m. where n=9 and the values represent the activity inhibited by 10 μM lactacystin. The differences from control are indicated as a, P<0.05 and b, P<0.001, while differences in the presence of EPA are indicated as c, P<0.05 and d, P<0.001.

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