Role of protein kinase C and NF-kappaB in proteolysis-inducing factor-induced proteasome expression in C(2)C(12) myotubes

Abstract

Proteolysis-inducing factor (PIF) is a sulphated glycoprotein produced by cachexia-inducing tumours, which initiates muscle protein degradation through an increased expression of the ubiquitin-proteasome proteolytic pathway. The role of kinase C (PKC) in PIF-induced proteasome expression has been studied in murine myotubes as a surrogate model of skeletal muscle. Proteasome expression induced by PIF was attenuated by 4alpha-phorbol 12-myristate 13-acetate (100 nM) and by the PKC inhibitors Ro31-8220 (10 microM), staurosporine (300 nM), calphostin C (300 nM) and Gö 6976 (200 microM). Proteolysis-inducing factor-induced activation of PKC(alpha), with translocation from the cytosol to the membrane at the same concentration as that inducing proteasome expression, and this effect was attenuated by calphostin C. Myotubes transfected with a constitutively active PKC(alpha) (pCO(2)) showed increased expression of proteasome activity, and a longer time course, compared with their wild-type counterparts. In contrast, myotubes transfected with a dominant-negative PKC(alpha) (pKS1), which showed no activation of PKC(alpha) in response to PIF, exhibited no increase in proteasome activity at any time point. Proteolysis-inducing factor-induced proteasome expression has been suggested to involve the transcription factor nuclear factor-kappaB (NF-kappaB), which may be activated through PKC. Proteolysis-inducing factor induced a decrease in cytosolic I-kappaBalpha and an increase in nuclear binding of NF-kappaB in pCO(2), but not in pKS1, and the effect in wild-type cells was attenuated by calphostin C, confirming that it was mediated through PKC. This suggests that PKC may be involved in the phosphorylation and degradation of I-kappaBalpha, induced by PIF, necessary for the release of NF-kappaB from its inactive cytosolic complex.

Figure 1

(A) Effect of PMA on PIF-induced chymotrypsin-like enzyme activity in murine myotubes. Cells were incubated either with PIF alone (×) or in the presence of PMA (100 nM), added 2 h prior to PIF (▪), and the chymotrypsin-like enzyme activity was determined after 24 h, as described in Materials and methods. The experiment was repeated three times (n=9). Differences from control are indicated as ^aP<0.001, while differences from cells incubated in the presence of PIF alone are shown as ^bP<0.05 and ^cP<0.001. (B) Western blot of the effect of PMA on proteasome 20S α-subunits and (C) E2_14k 24 h after addition of PIF. Cells were incubated with 0 (lanes 1 and 7), 1.0 (lanes 2 and 8), 2.1 (lanes 3 and 9), 4.2 (lanes 4 and 10), 10 (lanes 5 and 11) or 20 nM PIF (lanes 6 and 12) in the absence (lanes 1–6) or presence (lanes 7–12) of PMA (100 nM). A representative blot is shown and the experiment was repeated three times.

Figure 2

Effect of inhibitors of PKC on PIF-induced chymotrypsin-like enzyme activity. Myotubes were incubated with PIF alone (×) or with Ro 31-8220 (1 μM) (A); staurosporine (300 nM) (B); calphostin C (300 nM) (C); or with Go 6976 (200 μM) (D) added 2 h prior to PIF, and chymotrypsin-like enzyme activity was determined 24 h after addition of PIF. The experiment was repeated three times (n=9). Differences from control are indicated as ^aP<0.05 and ^bP<0.001, while differences from cells incubated in the presence of PIF alone are shown as ^cP<0.05, ^dP<0.01 and ^eP<0.001.

Figure 3

Western blot of the effect of calphostin C on 20S proteasome α-subunit expression (A) and E2_14k (B) in the presence of PIF. Cells were incubated with 0 (lanes 1 and 6), 2.1 (lanes 2 and 7), 4.2 (lanes 3 and 8), 10 (lanes 4 and 9) or 16.8 nM PIF (lanes 5 and 10) either alone (lanes 1–5) or in the presence of calphostin C (300 nM) and expression was determined after 24 h. A representative blot is shown and the densitometric analysis is based on three replicate blots. Values in the presence of PIF are indicated as ▪ and in the presence of PIF and calphostin C as □. The densitometric analysis of the 20S subunits represents the average of the two major bands. Differences from control are indicated as ^aP<0.05, ^bP<0.01 and ^cP<0.001, while differences from the presence of PIF alone are shown as ^dP<0.05, ^eP<0.01 and ^fP<0.001.

Figure 4

Effect of PIF on activation of PKC_α in murine myotubes in the absence or presence of calphostin C (A, B) or EPA (C). (A) Cytoplasmic and (B) Membrane-bound PKC_α after incubation with 0 (lanes 1 and 6), 2.1 (lanes 2 and 7), 4.2 (lanes 3 and 8), 10 (lanes 4 and 9) or 16.8 nM PIF (lanes 5 and 10) for 24 h in the absence (lanes 1–5) or presence (lanes 6–10) of calphostin C (300 nM). The densitometric analysis was based on three replicate blots, and values in the presence of PIF are shown as ▪ and in the presence of PIF and calphostin C as □. Differences from control are shown as ^cP<0.001, while differences from PIF alone are shown as ^dP<0.05, ^eP<0.01 and ^fP<0.001. (C) Actin loading control for the blots shown in (A, B). (C) Actin loading control for the blots shown in (A, B). (D) Effect of EPA (50 μM) on membrane-bound PKC_α in the presence of PIF. Cells were loaded with 0 (lanes 1 and 7), 1.0 (lanes 2 and 8), 2.1 (lanes 3 and 9), 4.2 (lanes 4 and 10), 10 (lanes 5 and 11) or 20 nM PIF (lanes 6 and 12) either in the absence (lanes 1–6) or after 2 h pretreatment with EPA (50 μM), and membrane-bound PKC_α was determined after 24 h. (E) β-tubulin loading control for the blot shown in (D).

Figure 5

Time course for induction of chymotrypsin-like enzyme activity in wild-type myotubes (×) and in those transfected with constitutively active (pCO₂ □) and mutant (pKS1 ▪) PKC_α. Myotubes were treated with the indicated concentrations of PIF, and enzyme activity was determined at 3 h (A), 6 h (B), 24 h (C) and 48 h (D). The experiment was repeated three times (n=9). Differences from control or pKS1 are indicated as ^aP<0.05, ^bP<0.01 and ^cP<0.005, while differences from wild-type myotubes are indicated as ^dP<0.005.

Figure 6

Western blot of the effect of PIF on 20S proteasome α-subunit expression (A) and E2_14k (B) in pKS1 (lanes 1–6) and pCO₂ (lanes 7–12) after treatment with 0 (lanes 1 and 7), 1.0 (lanes 2 and 8), 2.1 (lanes 3 and 9), 4.2 (lanes 4 and 10), 10 (lanes 5 and 11) and 20 nM PIF (lanes 6 and 12) determined after 24 h. (C) β-tubulin loading control.

Figure 7

Western blot of the effect of PIF on cytoplasmic (A) and membrane-bound (B) PKC_α in pKS1 and pCO₂ after 24 h. The lanes are the same as in Figure 6.

Figure 8

Effect of PIF and calphostin C on cytoplasmic I-κBα (A) and nuclear-bound NF-κB (C) determined 30 min after PIF addition to murine myotubes. (A) Myotubes were treated with 0 (lanes 1 and 6), 2.1 (lanes 2 and 7), 4.2 (lanes 3 and 8), 10 (lanes 4 and 9) or 16.8 nM PIF (lanes 5 and 10) in the absence (lanes 1–5) or presence (lanes 6–10) of calphostin C (300 nM) added 2 h prior to PIF. (B) Actin loading control for the blot shown in (A). (C) Nuclear levels of NF-κB in myotubes in the absence (lanes 2–6) or presence (lanes 7–11) of calphostin C. Lane 1 is a negative control and lane 13 a positive control and lane 12 contains excess unlabelled NF-κB. The other lanes were the same as in (A). The densitometric analysis is an average of three replicate EMSAs. Differences from control are indicated as ^aP<0.05 and ^cP<0.001, while differences in the presence of calphostin C are indicated as ^dP<0.01 and ^eP<0.001.

Figure 9

Effect of PIF on cytoplasmic I-κBα (A) and nuclear-bound NF-κB (C) determined 30 min after PIF addition to murine myotubes transfected with constitutively active (pCO₂ □) and mutant (pKS1 ▪) PKC_α. (A) Myotubes transfected with either pCO₂ (lanes 1–6) or pKS1 (lanes 7–12) were treated with 0 (lanes 1 and 7), 1.05 (lanes 2 and 8), 2.1 (lanes 3 and 9), 4.2 (lanes 4 and 10), 10 (lanes 5 and 11) or 16.8 nM PIF (lanes 6 and 12). The densitometric analysis is an average of three replicate blots. Differences from 0 nM PIF are shown as ^cP<0.001. (B) Actin loading control for the blot shown in (A). (C) EMSA of NF-κB nuclear binding in murine myotubes transfected with pCO₂ (lanes 2 and 6) and pK1 (lanes 7–11). Myotubes were treated with 0 (lanes 2 and 7), 2.1 (lanes 3 and 8), 4.2 (lanes 4 and 9), 10 (lanes 5 and 10) and 16.8 nM PIF (lanes 6 and 11). Lane 1 is a negative control containing the labelled probe without a nuclear extract; lane 12 contains a 100-fold excess of unlabelled NF-κB probe and lane 13 is a positive control for NF-κB (supplied by the manufacturers of the kit). The densitometric analysis is an average of three replicate EMSAs. Differences from control are indicated as ^bP<0.01 and ^cP<0.001.