NF-kappaB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin-proteasome system in skeletal muscle

Abstract

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-kappaB (NF-kappaB) in regulating muscle protein degradation and expression of the ubiquitin-proteasome proteolytic pathway in response to a tumour cachectic factor, proteolysis-inducing factor (PIF), has been studied by creating stable, transdominant-negative, muscle cell lines. Murine C(2)C(12) myoblasts were transfected with plasmids with a CMV promoter that had mutations at the serine phosphorylation sites required for degradation of I-kappaBalpha, an NF-kappaB inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-kappaBalpha, nuclear accumulation of NF-kappaB and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-kappaBalpha proteins. Proteolysis-inducing factor also induced total protein degradation and loss of the myofibrillar protein myosin in myotubes containing wild-type, but not mutant, plasmids at the same concentrations as those causing activation of NF-kappaB. Proteolysis-inducing factor also induced increased expression of the ubiquitin-proteasome pathway, as determined by 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the beta-subunits of the proteasome, protein expression of 20S alpha-subunits and the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme, E2(14k), in cells containing wild-type, but not mutant, I-kappaBalpha. The ability of mutant I-kappaBalpha to inhibit PIF-induced protein degradation, as well as expression of the ubiquitin-proteasome pathway, confirms that both of these responses depend on initiation of transcription by NF-kappaB.

Figure 1

Effect of mutation on degradation of I-κBα in the presence of PIF. (A) Western blot analysis of I-κBα after 30 min incubation with 0 (lanes 1, 6 and 11), 2.1 (lanes 2, 7 and 12), 4.2 (lanes 3, 8 and 13), 10.5 (lanes 4, 9 and 14) and 16.8 (lanes 5, 10 and 15) nM PIF in wild-type cells transfected with pCMV4 (lanes 1–5), I-κBαS32/A36 (lanes 6–10) and I-κBαΔN (lanes 11–15). (B) Densitometric analysis of the blot shown in (A) as the mean±s.e.m. for three separate determinations. Differences from control are indicated as c, P<0.001, while differences from wild-type are shown as f, P<0.001. (C) Western blot of actin showing equal loading of samples in (A).

Figure 2

Activation of NF-κB binding to DNA as demonstrated by EMSA. Only the band for bound NF-κB is shown. C₂C₁₂ myotubes were treated with 0 (lanes 1 and 6), 2.1 (lanes 2 and 7), 4.2 (lanes 3 and 8), 10.5 (lanes 4 and 9) and 16.8 (lanes 5 and 10) nM PIF. Binding of NF-κB to nuclear proteins was determined in cells transfected with pCMV4 (lanes 1–5) and I-κBα S32/A36 (lanes 6–10) (A) and in pCMV4 (lanes 1–5) and I-κBαΔN (lanes 6–10) (B). Lane 12 is a positive control for NF-κB (supplied by the manufacturer of the kit), while lane 11 contains the positive control for NF-κB together with a 100-fold excess of unlabelled NF-κB probe. The densitometric analysis of the blots is shown underneath the EMSA. Wild type is shown as solid boxes, while the mutants are shown as open boxes. Figures are means±s.e.m. for three separate determinations. Differences from control are indicated as a, P<0.05 and c, P<0.001, while differences from wild-type are shown as f, P<0.001.

Figure 3

Effect of PIF on the transcriptional activity of NF-κB measured by the luciferase reporter gene assay in myotubes transfected with pCMV4 (▪), S32/A36 () and I-κBΔN (□) after 1 h incubation. Sample luciferase activity was normalised to control luciferase activity. The results shown are mean±s.e.m., where n=3. Differences from 0 nM PIF for pCMV4 is shown as a, P<0.05 or c, P<0.001, while differences from wild-type myotubes are indicated as e, P<0.01 or f, P<0.001.

Figure 4

Effect of PIF on total protein degradation in myotubes transfected with pCMV4 (▪), I-κBα S32/A36 () and I-κBαΔN (□) over a 24 h period. Results are shown as mean±s.e.m. of one experiment, where n=6, and the experiment was repeated twice on different days with similar results. Differences from 0 nM PIF for pCMV4 is shown as c, P<0.001, while differences between I-κBα S32/A36 and I-κBαΔN and pCMV4 are shown as e, P<0.01 and f, P<0.001.

Figure 5

Effect of PIF on myosin content of myotubes containing wild-type and mutant I-κBα. (A) Wild-type myotubes transfected with pCMV4 (lanes 1–5), I-κBα S32/A36 (lanes 6–10) and I-κBαΔN (lanes 11–15) myotubes were incubated with 0 (lanes 1, 6 and 11), 2.1 (lanes 2, 7 and 12), 4.2 (lanes 3, 8 and 13), 10.5 (lanes 4, 9 and 14) or 16.8 (lanes 5, 10 and 15) nM PIF for 24 h and myosin content was determined by Western blotting. (B) Densitometric analysis of the blot shown in (A). Figures are mean±s.e.m. of three separate determinations. Differences from 0 nM PIF are shown as a, P<0.05 and c, P<0.001, while differences from wild-type myotubes are indicated as f, P<0.001. (C) Western blot of actin showing equal loading in (A).

Figure 6

The effect of mutation of I-κBα on the chymotrypsin-like enzyme activity in murine myotubes after treatment with PIF. pCMV4 myotubes (▪); I-κBα S32/A36 (□) and I-κBαΔN() were treated with the indicated concentrations of PIF for 24 h and the ‘chymotrypsin-like’ enzyme activity was determined fluorimetrically as described in Materials and Methods. Differences from 0 nM PIF for pCMV4 are indicated as c, P<0.001, while differences from wild-type myotubes are indicated as f, P<0.001.

Figure 7

(A) Effect of PIF on 20S proteasome α-subunit expression in myotubes transfected with pCMV4 (lanes 1–5), I-κBα S32/A36 (lanes 6–10) and I-κBαΔN (lanes 11–15) plasmids. Myotubes were incubated for 24 h with 0 (lanes 1, 6 and 11), 2.1 (lanes 2, 7 and 12), 4.2 (lanes 3, 8 and 13), 10.5 (lanes 4, 9 and 14) and 16.8 (lanes 5, 10 and 15) nM PIF and proteasome expression was determined by Western blotting of 5 μg of cytosolic protein. (B) Densitometric analysis of three replicate blots as shown in (A) ▪ band 1; □ band 2. Differences from 0 nM PIF are indicated as a, P<0.05, b, P<0.01 and c, P<0.001, while differences from wild-type myotubes are indicated as d, P<0.05 and f, P<0.001. (C) Western blot of actin from the blot shown in (A).

Figure 8

(A) Effect of PIF on MSS1 expression in myotubes transfected with wild type (lanes 1–5), I-κBα S32/A36 (lanes 6–10) and I-κBαΔN (lanes 11–15) plasmids. Myotubes were incubated for 24 h with 0 (lanes 1, 6 and 11), 2.1 (lanes 2, 7 and 12), 4.2 (lanes 3, 8 and 13), 10.5 (lanes 4, 9 and 14) and 16.8 (lanes 5, 10 and 15) nM PIF and MSS1 expression was determined by Western blotting of 5 μg of cytosolic protein. (B) Densitometric analysis of three replicate blots shown in (A). Differences from 0 nM PIF are indicated as c, P<0.001, while differences from wild-type controls are shown as d, P<0.05 and f, P<0.001. (C) Western blot of actin from the blot shown in (A).

Figure 9

(A) Effect of PIF on p42 expression in myotubes transfected with pCMV4 (lanes 1–5), I-κBα S32/A36 (lanes 6–10) and I-κBαΔN (lanes 11–15) plasmids. Myotubes were incubated for 24 h with 0 (lanes 1, 6 and 11), 2.1 (lanes 2, 7 and 12), 4.2 (lanes 3, 8 and 13), 10.5 (lanes 4, 9 and 14) and 16.8 (lanes 5, 10 and 15) nM PIF and p42 expression was determined by western blotting. (B) Densitometric analysis of three replicate blots shown in (A). Differences from 0 nM PIF are shown as c, P<0.001, while differences from wild-type controls are shown as f, P<0.001. (C) Western blot of actin from the blot shown in (A).

Figure 10

Effect of PIF on E2_14k expression in myotubes transfected with pCMV4 (lanes 1–5), I-κBα S32/A36 (lanes 6–10) and I-κBαΔN (lanes 11–15) plasmids. Myotubes were incubated for 24 h with 0 (lanes 1, 6 and 11), 2.1 (lanes 2, 7 and 12), 4.2 (lanes 3, 8 and 13), 10.5 (lanes 4, 9 and 14) and 16.8 (lanes 5, 10 and 15) nM PIF and p42 expression was determined by Western blotting. (B) Densitometric analysis of three replicate blots shown in (A). Differences from 0 nM PIF are shown as c, P<0.001, while differences from wild-type controls are shown as f, P<0.001. (C) Western blot of actin from the blot shown in (A).