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

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.

Figure 5

(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.

Figure 1

Effect of PIF in the absence (black boxes) or presence (grey boxes) of lactacystin (10 μM) on protein degradation in C₂C₁₂ myotubes as determined by [³H] phenylalanine release. Lactacystin was present for 2 h prior to PIF and was also present throughout the 24 h incubation period. All values have been normalised to their respective controls and the experiment was repeated twice, n=3 for each treatment. The data are expressed as mean±s.e.m. and was measured 24 h after the addition of PIF. Differences from control are indicated as a, P<0.01 and b, P<0.001, while differences in the presence of lactacystin are shown as c, P<0.001.

Figure 2

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.

Figure 3

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.

Figure 4

(A) The effect of PIF on IκBα expression in C₂C₁₂ myotubes at 30, 60 and 120 min as determined by Western blotting. Lanes 1, 2 and 3 represent 0, 4 and 20 nM PIF after 30 min; lanes 4–6 after 60 min and lanes 7–9 after 120 min. (B) Densitometric analysis of the blot shown in (A) n=2. Differences from control are shown as a, P<0.001. (C) The effect of PIF on IκBα expression in C₂C₁₂ myotubes after 30 min in the absence (lanes 1–5) or presence (lanes 6–10) of 50 μM EPA added 2 h prior to PIF. The lanes represent the following concentrations of PIF: 1 and 6, 0 nM; 2 and 7, 2 nM; 3 and 8, 4 nM; 4 and 9, 8 nM and 5 and 10, 20 nM. (D) Densitometric analysis of the blot shown in (C), n=2. Differences from control are shown as a, P<0.001.