In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser473 within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin–proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin–proteasome pathway through activation of NF-κB (nuclear factor κB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-κB and that this also allows for a specific synthesis of proteasome subunits.

INTRODUCTION

The ubiquitin–proteasome proteolytic pathway has been shown to play a major role in muscle wasting in starvation, sepsis, metabolic acidosis, diabetes, weightlessness, severe trauma, denervation and cancer cachexia [1]. This pathway can be activated by a number of factors, including PIF (proteolysis-inducing factor), a sulfated glycoprotein produced by cachexia-inducing tumours [2], cytokines such as TNFα (tumour necrosis factor α) and IFN-γ (interferon-γ) [3], Ang II (angiotensin II) [4] and glucocorticoids [5], whereas disuse atrophy results from an increase in oxidative stress [6]. Induction of protein degradation through the ubiquitin–proteasome pathway by PIF [7], TNFα [8], Ang II [9] and ROS (reactive oxygen species) [10] involves activation of the nuclear transcription factor NF-κB (nuclear factor κB). Activation of NF-κB through muscle-specific transgenic expression of activated IKKβ [IκB (inhibitory κB) kinase β] has been shown [11] to induce profound muscle wasting with an increase in mRNA levels for the C2 and C9 subunits of the proteasome, as well as for MuRF1, a muscle-specific E3 ligase, which is up-regulated during atrophy, and loss of which provides partial protection (36%) from muscle loss after denervation [12]. Interestingly mRNA levels for another E3 ligase atrogin-1/MAFbx, also implicated in muscle protein degradation [13], were normal, as were those for ubiquitin and the ubiquitin-conjugating enzyme E214k. Induction of atrogin-1 has been shown to be mediated by the Foxo (Forkhead box O) class of transcription factors [14], suggesting that there may be two distinct mechanisms for the induction of muscle atrophy.

Recent studies [15] on cellular signalling pathways involved in the induction of muscle protein degradation by both PIF and Ang II indicate that a transitory rise in ROS is an important event leading to up-regulation of the ubiquitin–proteasome pathway through activation of NF-κB, through an increased phosphorylation and degradation of the inhibitor protein IκBα. These studies showed that ROS formation required the stimulation of PI3K (phosphoinositide 3-kinase), which is known to activate the small GTP-binding protein Rac1 [16]. Also the lipid product of PI3K, phosphoinositide 3-phosphate, forms a scaffold for membrane attachment of the NADPH oxidase components p40phox and p47phox [17]. Thus treatment with the highly selective PI3K inhibitor LY294002 [18] completely attenuated both ROS production and protein degradation induced by PIF and Ang II, whereas the structural analogue LY303511, containing a single atom substitution in the morpholine ring, and which is devoid of PI3K inhibitory activity, had no effect on either ROS formation or protein degradation.

Both growth factors and cytokines also activate the serine/threonine protein kinase, Akt/PKB (protein kinase B) via a PI3K pathway. Oxidative stress has also been shown to activate Akt by increasing phosphorylation [19]. In vascular smooth muscle cells [20] and in hypertrophic scar fibroblasts [21] Ang II has been shown to induce the PI3K activation of Akt. Activation of Akt could provide an alternative pathway for increased nuclear binding of NF-κB through phosphorylation and activation of the upstream kinase IKK. This pathway is utilized by both TNFα [22] and PDGF (platelet-derived growth factor) [23] to activate NF-κB. However, stimulation of the PI3K/Akt pathway by IGF-I (insulin-like growth factor I) has been shown to induce hypertrophy [24], whereas constitutive activation of Akt induces rapid and significant skeletal muscle hypertrophy [25]. The hypertrophic effect is due to stimulation of translation, via regulation of GSK (glycogen synthase kinase) and mTOR (mammalian target of rapamycin) kinases [26]. Akt overexpression inhibits the Foxo transcription factors by increasing phosphorylation, inhibiting atrogin-1 expression [14].

These apparently conflicting roles for Akt led us to investigate whether activation of Akt did occur during protein degradation in muscle, and if so what this role was. To do this we have transfected myotubes with plasmids expressing a dominant-negative mutant of Akt (termed DNAkt), as well as a constitutively active Akt construct (termed MyrAkt), and studied the effect on both protein degradation and proteasome activity in response to PIF. Using an inhibitor of PI3K, and the dominant-negative and constitutively active Akt constructs, the effects have been associated with the state of activation of NF-κB, as well as a transient stimulation of protein synthesis through the mTOR and the p70S6k (70 kDa ribosomal protein S6 kinase) pathways. This is possibly required for synthesis of proteasome subunits, as well as for the synthesis of the E3 ligases, MuRF1 and atrogin-1/MAFbx, the synthesis of which are increased prior to the initiation of degradation of myofibrillar proteins.

MATERIALS AND METHODS

FCS (foetal calf serum), HS (horse serum) and DMEM (Dulbecco's modified Eagle's medium) were purchased from Life Technologies. A mouse monoclonal antibody to IκBα was purchased from Biomol Research Laboratories and a monoclonal antibody to HA.11 from Cambridge Bioscience. Mouse monoclonal antibodies to 20S proteasome α-subunits and p42 were from Affiniti Research Products. Mouse monoclonal antibody to myosin heavy chain was from Novocastra. Rabbit polyclonal antisera to phosphorylated IκBα and to phosphorylated GSK3α/β (phospho Tyr279/216), as well as Phosphosafe™ extraction reagent were from Merck Biosciences. Polyclonal antisera to Akt that detects endogenous Akt1, Akt2 and Akt3 proteins, phosphorylated mTOR, phosphorylated p70S6k, and their unphosphorylated forms, together with a mouse monoclonal antibody to phospho-Akt (Ser473) were from New England Biolabs. Rabbit polyclonal antisera to mouse actin was from Sigma–Aldridge. Peroxidase-conjugated rabbit anti-mouse antibody and peroxidase-conjugated goat anti-rabbit antibody were purchased from Dako. Hybond A nitrocellulose membranes, L-[2,6−3H]phenylalanine (specific radioactivity, 2.07 TBq/mmol) and ECL® (enhanced chemiluminescence) development kits were from Amersham Bioscience. LY294002 was purchased from Calbiochem (through CN Biosciences). Lactacystin was purchased from Affiniti Research Products and the chymotrypsin substrate succinyl-Leu-Leu-Val-7-AMC (where AMC is amino-methyl coumarin) from Sigma–Aldridge. Expression vectors (pcDNA) encoding mouse Akt proteins fused in-frame to the HA (haemagglutinin) epitope [26] were kindly supplied by Dr Kenneth Walsh (Tufts University, School of Medicine, Boston, U.S.A.). Catch and Release version 2.0 reversible immunoprecipitation system was purchased from Upstate. This system was used to immunoprecipitate Akt from transfected myotubes using the HA.11 monoclonal antibody according to the manufacturer's instructions.

Cell culture

The C2C12 myoblast cell line was grown in DMEM supplemented with 10% FCS plus 1% penicillin and streptomycin under an atmosphere of 5% CO2 in air at 37 °C. Cells were transfected with the plasmid DNA in OPTI-medium using Lipofectamine™ (Invitrogen) at a ratio of 1:5 and incubated at 37 °C for 18–48 h prior to testing for transgene expression. Cells were passaged (1:10) in fresh growth medium for 24 h and selected for growth in neomycin for 48–72 h. Myotubes were formed by allowing confluent cultures of myoblasts to fuse in DMEM containing 2% HS over a 5–7 day period, with changes of medium every 2 days.

Purification of PIF

PIF was purified from solid MAC16 tumours from mice with weight loss of between 20 and 25%. All animal experiments followed a strict protocol approved by the British Home Office. The tumour homogenate was precipitated with ammonium sulfate (40% w/v), and the supernatant was subjected to chromatography using a monoclonal antibody immobilized to a protein A matrix as described previously [27]. The immunogenic fractions were concentrated and used for further studies. As previously reported [27] these contained albumin as well as PIF, but there were no other contaminants. The albumin co-purifies with PIF because it binds PIF strongly [28], and this is difficult to remove, except by HPLC, but the resulting product may be contaminated with solvents that are toxic to the myotubes.

Measurement of protein degradation

The method for the measurement of protein degradation in murine myotubes has been described previously [2]. Briefly myotubes were prelabelled for 24 h with L-[2,6−3H]phenylalanine, and were washed extensively, followed by a 2 h incubation, to allow degradation of short-lived proteins prior to experimentation. Protein degradation was determined by the release of L-[2,6−3H]phenylalanine into the medium after 24 h incubation with various concentrations of PIF, Ang I or Ang II as depicted in the Figure legends in the absence or presence of inhibitors added 2 h before the agonists. Non-radioactive phenylalanine (2 mM) was added to prevent reincorporation of radioactivity into cells.

Measurement of proteasome ‘chymotrypsin-like’ activity

‘Chymotrypsin-like’ enzyme activity was determined fluorimetrically by the method of Orino et al. [29] as previously described [7]. Myotubes were washed with ice-cold PBS and sonicated in 20 mM Tris/HCl (pH 7.5), 2 mM ATP, 5 mM MgCl2 and 1 mM dithiothreitol at 4 °C. The supernatant formed by centrifugation at 15000 g for 10 min was used to measure ‘chymotrypsin-like’ enzyme activity by the release of AMC from the fluorogenic peptide succinyl-LLVY-AMC (0.1 mM). Activity was measured in the presence and absence of the specific proteasome inhibitor lactacystin (10 μM). Of the total activity, 60% was suppressible with lactacystin. Only lactacystin-suppressible activity was considered to be proteasome specific.

Measurement of protein synthesis

Myotubes were formed in 6-well multiwell dishes, and were supplemented with DMEM without HS and Phenol Red 18 h prior to experimentation. PIF was added at the concentrations indicated followed by 2 μl of L-[2,6-3H]phenylalanine (specific radioactivity, 1.96 TBq/mmol) in 8 μl sterile PBS and the plates were incubated for 4 h at 37 °C under an atmosphere of 5% CO2 in air. The reaction was arrested by washing three times with 1 ml of ice-cold sterile PBS. Following removal of the PBS 1 ml of ice-cold 0.2 M perchloric acid was added and the plates were kept at 4 °C for 20 min. The perchloric acid was substituted with 1 ml of 0.3 M NaOH per well and incubation was continued for 30 min at 4 °C, followed by a further incubation at 37 °C for a further 20 min. The NaOH extract was removed and combined with a further 1 ml wash of each well and 0.5 ml of 0.2 M perchloric acid was added and left on ice for 20 min. The extract was then centrifuged at 700 g for 5 min at 4 °C and the protein-containing pellet was dissolved in 1 ml of 0.3 M NaOH and 0.5 ml of the solution was counted for radioactivity after mixing with 8 ml of Ultima Gold XR scintillation fluid.

Western blot analysis

Cytoplasmic proteins (5–15μg), obtained from the chymotrypsin assay, were also used for Western blotting 20S proteasome α-subunits, p42 and myosin. For IκBα, phospho-IκBα, Akt, p70S6k and mTOR, samples were extracted with Phosphosafe™ Extraction Reagent. A 5 μg aliquot of immunoprecipitated protein from myotubes using HA.11 monoclonal antibody was used for immunoblotting for Akt. Proteins were resolved on SDS/PAGE (12% gels) and transferred to Hybond™ nitrocellulose membrane. Membranes were blocked with 5% non-fat dried skimmed milk (Marvel) in PBS for 1–2 h at room temperature (18–20 °C) and incubated with the primary antibodies at 4 °C overnight, except for p42 and 20S where incubation was for 1–2 h at room temperature. The primary antibodies were used at a dilution of 1:1000, except for myosin, which was used at a dilution of 1:40, and the secondary antibodies were also used at a dilution of 1:1000. Incubation was carried out for 2 h at room temperature, and development was by ECL. Total cellular actin was used as a loading control. Blots were scanned using a densitometer to quantify differences.

EMSA (electrophoretic mobility-shift assay)

DNA-binding proteins were extracted from myotubes by the method of Andrews and Faller [30], which utilizes hypotonic lysis followed by high-salt extraction of nuclei. The EMSA-binding assay was carried out using a Panomics EMSA ‘gel-shift’ kit according to the manufacturer's instructions.

Statistical analysis

Differences in means between groups was determined by one-way ANOVA followed by the Tukey–Kramer Multiple Comparison Test. All experiments were repeated at least three times on separate occasions and the results shown are an average of three repeats.

RESULTS

PIF induced a transient increase in Akt phosphorylation at Ser473 in murine myotubes, while having no effect on the total Akt in the cell (Figure 1A). Akt phosphorylation was increased 4-fold within 30 min of addition and rapidly returned to baseline values. This time corresponds with the time of maximal ROS formation, when PI3K would be activated [15]. This was confirmed by co-treatment with the PI3K inhibitor LY294002 [18], which completely inhibited induction of Akt phosphorylation by PIF (Figure 1B). Akt phosphorylation by PIF was also completely inhibited by the tyrosine kinase inhibitor genistein (Figure 1C), suggesting that activation of Akt occurs through a similar mechanism to that induced by growth factors and cytokines.

Effect of PIF on phosphorylation of Akt

Figure 1
Effect of PIF on phosphorylation of Akt

(A) Western blot analysis of the time course of PIF (4.2 nM) on phosphorylation of Akt at Ser473 in murine myotubes, maintained in unsupplemented DMEM without Phenol Red for 18 h prior to experimentation. A densitometric analysis representing the average of three separate blots is shown below the Western blot. (B) Western blot analysis of the effect of PIF concentration on phosphorylation of Akt, in the absence or presence of LY294002. Myotubes were treated with PIF for 30 min in the absence or presence of 25 μM LY294002 added 2 h prior to the addition of PIF. (C) Western blot analysis of the effect of PIF concentration on phosphorylation of Akt after 30 min in the absence or presence of 30 μM genistein added 2 h prior to the addition of PIF. The blots shown are representative of three separate experiments and total Akt was used as a loading control. The densitometric analysis is the mean of the three separate Western blots and shows the intensity of the phospho-Akt signal in the presence of PIF alone (solid bars) or in the presence of LY294002 (B) or genistein (C). Differences from control in the absence of PIF are shown as b, P<0.01 or c, P<0.001, whereas differences in the presence of LY294002 or genestein are indicated as f, P<0.001. There was no difference in the level of total Akt in the cells for any of the treatments.

Figure 1
Effect of PIF on phosphorylation of Akt

(A) Western blot analysis of the time course of PIF (4.2 nM) on phosphorylation of Akt at Ser473 in murine myotubes, maintained in unsupplemented DMEM without Phenol Red for 18 h prior to experimentation. A densitometric analysis representing the average of three separate blots is shown below the Western blot. (B) Western blot analysis of the effect of PIF concentration on phosphorylation of Akt, in the absence or presence of LY294002. Myotubes were treated with PIF for 30 min in the absence or presence of 25 μM LY294002 added 2 h prior to the addition of PIF. (C) Western blot analysis of the effect of PIF concentration on phosphorylation of Akt after 30 min in the absence or presence of 30 μM genistein added 2 h prior to the addition of PIF. The blots shown are representative of three separate experiments and total Akt was used as a loading control. The densitometric analysis is the mean of the three separate Western blots and shows the intensity of the phospho-Akt signal in the presence of PIF alone (solid bars) or in the presence of LY294002 (B) or genistein (C). Differences from control in the absence of PIF are shown as b, P<0.01 or c, P<0.001, whereas differences in the presence of LY294002 or genestein are indicated as f, P<0.001. There was no difference in the level of total Akt in the cells for any of the treatments.

Activation of Akt is associated with stimulation of protein synthesis in muscle [24]. However, PIF is known to inhibit protein synthesis through activation of the PKR (double-stranded-RNA-dependent protein kinase) and the subsequent phosphorylation of eIF2α (eukaryotic initiation factor 2α) [31]. However, protein synthesis is required during protein degradation for the synthesis of proteasome subunits, ubiquitin and ubiquitin ligases for which mRNA levels are known to be elevated [12]. Activation of Akt causes activation of mTOR, which has two primary downstream targets, p70S6k and the eIF-4E (eukaryotic initiation factor 4E)-binding protein, phosphorylation of which leads to accelerated protein synthesis [32]. To determine whether this pathway was important in protein degradation, myotubes were treated with the mTOR inhibitor rapamycin (25 ng/ml), either before or at various times after, the addition of PIF (Figure 2A). Addition of rapamycin either 2 h before or up to 0.5 h after addition of PIF completely attenuated protein degradation, whereas addition at later times had no effect (Figure 2A). Addition of rapamycin also attenuated induction of the ubiquitin–proteasome pathway, as determined by the ‘chymotrypsin-like’ enzyme activity (Figure 2B) with a time course the same as that for total protein degradation (Figure 2C). This time course also correlated with the induction of phosphorylation of mTOR by PIF (Figure 2C). As with the time course for phosphorylation of Akt (Figure 1A), and the inhibition of protein degradation by rapamycin, the effect of PIF on mTOR phosphorylation was maximal at 30 min (Figure 2C), and was not significantly elevated after 1 h. Activation of mTOR was followed by induction of phosphorylation of p70S6k, which was maximal between 1 and 2 h after addition of PIF (Figure 2D). These results suggest that the PI3K/Akt/mTOR pathway may also be important for a short burst of protein synthesis producing proteasome subunits and ubiquitin ligases required for protein degradation through the ubiquitin–proteasome pathway.

Involvement of mTOR in cellular signalling by PIF

Figure 2
Involvement of mTOR in cellular signalling by PIF

(A) Time course for the effect of rapamycin (25 ng/ml) on total protein degradation induced by PIF (4.2 nM) over a 24 h period. (B) Time course for the effect of rapamycin on proteasome ‘chymotrypsin-like’ enzyme activity in the presence of PIF (4.2 nM) added at zero time, and measured after a further 24 h period. The values for PIF alone are shown as solid bars whereas the values in the presence of PIF together with rapamycin are shown as open bars. aP<0.05, bP<0.01 and cP<0.001 compared with controls; fP<0.001 compared with PIF alone. (C) Western blot analysis showing the time course for phosphorylation of mTOR by PIF. (D) Western blot analysis showing a time course for phosphorylation of p70S6k by PIF. A densitometric analysis representing the average of three separate Western blots is shown underneath. aP<0.05, bP<0.01 or cP<0.001 compared with control.

Figure 2
Involvement of mTOR in cellular signalling by PIF

(A) Time course for the effect of rapamycin (25 ng/ml) on total protein degradation induced by PIF (4.2 nM) over a 24 h period. (B) Time course for the effect of rapamycin on proteasome ‘chymotrypsin-like’ enzyme activity in the presence of PIF (4.2 nM) added at zero time, and measured after a further 24 h period. The values for PIF alone are shown as solid bars whereas the values in the presence of PIF together with rapamycin are shown as open bars. aP<0.05, bP<0.01 and cP<0.001 compared with controls; fP<0.001 compared with PIF alone. (C) Western blot analysis showing the time course for phosphorylation of mTOR by PIF. (D) Western blot analysis showing a time course for phosphorylation of p70S6k by PIF. A densitometric analysis representing the average of three separate Western blots is shown underneath. aP<0.05, bP<0.01 or cP<0.001 compared with control.

To confirm a role of Akt in protein degradation induced by PIF, murine myoblasts were transfected with plasmids encoding a dominant-negative Akt (termed DNAkt) in which the two activating amino acid residues, Thr308 and Ser473, were changed to an alanine residue [26], or a constitutively active Akt construct (termed MyrAkt), containing the c-Src myristoylation sequence, and allowed to differentiate into myotubes. Control myoblasts were transfected with the empty vector pcDNA3(−). Immunoprecipitation of cell lysates of myotubes formed by fusion of transfected myoblasts with an anti-HA antibody, followed by Western blotting for Akt, showed expression of Akt in myotubes, confirming that this was a stable transfection (Figure 3A). The level of expression of transfected HA–Akt relative to the endogenous protein is shown in Figure 3(B), which confirms the lack of expression in myotubes containing empty plasmid [pcDNA3(−)]. To confirm that the plasmids were influencing cellular pathways as expected, myotubes were treated with IGF-I (13.2 nM) for 1 h and the levels of phosphorylation of p70S6k (Figure 3C) and GSK3β (Figure 3D) was determined. As expected IGF-I increased the level of phosphorylation of p70S6k and GSK3β in myotubes containing the empty plasmid [pcDNA3(−)], but not in those containing DNAkt. Myotubes containing MyrAkt showed an increased phosphorylation of p70S6k and GSK3β, even in the absence of IGF-I. These results confirm that the transfected plasmids were functional in terms of Akt activity.

Characterization of myotubes transfected with MyrAkt and DNAkt compared with empty plasmid

Figure 3
Characterization of myotubes transfected with MyrAkt and DNAkt compared with empty plasmid

(A) Western blot analysis of immunoprecipitated HA-tagged Akt in myotubes transfected with empty pcDNA plasmid (lane 1), MyrAkt (lane 2) and DNAkt (lane 3) using rabbit polyclonal antisera to Akt. The lower band is the Ig heavy-chain. The myoblasts had undergone three passages in culture after transfection before being differentiated into myotubes. (B) Level of expression of transfected HA-Akt relative to endogenous protein in myotubes transfected with pcDNA, MyrAkt and DNAkt, using rabbit polyclonal antisera to Akt. Each lane represents protein from a separate cell culture. (C) Western blot analysis of the effect of IGF-I (13.2 nM) on phosphorylation of p70S6k and on GSK3β (D) in myotubes transfected with pcDNA3(−), MyrAkt and DNAkt after 4 h treatment. An actin loading control is shown in the lower panel. The experiment was repeated three times and the densitometric analysis is the average of the three blots. aP<0.05, bP<0.01 and cP<0.001 compared with control; dP<0.05, eP<0.01 or fP<0.001 compared with pcDNA3(−).

Figure 3
Characterization of myotubes transfected with MyrAkt and DNAkt compared with empty plasmid

(A) Western blot analysis of immunoprecipitated HA-tagged Akt in myotubes transfected with empty pcDNA plasmid (lane 1), MyrAkt (lane 2) and DNAkt (lane 3) using rabbit polyclonal antisera to Akt. The lower band is the Ig heavy-chain. The myoblasts had undergone three passages in culture after transfection before being differentiated into myotubes. (B) Level of expression of transfected HA-Akt relative to endogenous protein in myotubes transfected with pcDNA, MyrAkt and DNAkt, using rabbit polyclonal antisera to Akt. Each lane represents protein from a separate cell culture. (C) Western blot analysis of the effect of IGF-I (13.2 nM) on phosphorylation of p70S6k and on GSK3β (D) in myotubes transfected with pcDNA3(−), MyrAkt and DNAkt after 4 h treatment. An actin loading control is shown in the lower panel. The experiment was repeated three times and the densitometric analysis is the average of the three blots. aP<0.05, bP<0.01 and cP<0.001 compared with control; dP<0.05, eP<0.01 or fP<0.001 compared with pcDNA3(−).

In order to determine the effect of Akt expression on protein degradation, myotubes were incubated with PIF and total protein degradation was measured over a 24 h period. In myotubes containing the empty plasmid, pcDNA3(−), PIF induced protein degradation with a bell-shaped dose–response curve, with a maximal effect at 4.2 nM, as previously reported [3,7,9,15,31] (Figure 4A). In contrast, myotubes containing MyrAkt showed an enhanced protein degradation over pcDNA3(−) at all concentrations of PIF, whereas those containing DNAkt showed a reduced protein degradation at the maximum activating concentration of PIF. The non-linear dose–response curve for PIF has been shown to directly relate to ROS formation [15] through activation of PKC (protein kinase C) [32], which is down-regulated at high concentrations of activating ligand. Protein degradation was significantly enhanced in myotubes expressing constitutively active Akt, whereas protein degradation was completely attenuated in myotubes expressing DNAkt, compared with those containing empty plasmid.

Role of Akt in protein degradation and induction of the ubiquitin–proteasome pathway by PIF

Figure 4
Role of Akt in protein degradation and induction of the ubiquitin–proteasome pathway by PIF

(A) Effect of various concentrations of PIF on total protein degradation and (B) on the 20S proteasome chymotrypsin-like enzyme activity in murine myotubes containing pcDNA3(−) (▲), MyrAkt (◆) or DNAkt (□). aP<0.05, bP<0.01 or cP<0.001 compared with control; dP<0.05, eP<0.01 or fP<0.001 compared with wild-type. All experiments were repeated at least three times. Expression of (C) 20S proteasome α-subunits, (D) β5-subunits, (E) p42, (F) myosin heavy chain and (G) p70S6k in myotubes containing pcDNA3(−) (solid bars), MyrAkt (stippled bars) and DNAkt (open bars) in response to PIF (4.2nM) after 4 h (p70S6k) or 24 h incubation. An actin loading control for (C), (D) and (E) is shown in (F). A densitometric analysis representing the average from three blots is shown below each Western blot. Differences from control are shown as cP<0.001 compared with control; dP<0.05, eP<0.01 or fP<0.001 compared with pcDNA3(−).

Figure 4
Role of Akt in protein degradation and induction of the ubiquitin–proteasome pathway by PIF

(A) Effect of various concentrations of PIF on total protein degradation and (B) on the 20S proteasome chymotrypsin-like enzyme activity in murine myotubes containing pcDNA3(−) (▲), MyrAkt (◆) or DNAkt (□). aP<0.05, bP<0.01 or cP<0.001 compared with control; dP<0.05, eP<0.01 or fP<0.001 compared with wild-type. All experiments were repeated at least three times. Expression of (C) 20S proteasome α-subunits, (D) β5-subunits, (E) p42, (F) myosin heavy chain and (G) p70S6k in myotubes containing pcDNA3(−) (solid bars), MyrAkt (stippled bars) and DNAkt (open bars) in response to PIF (4.2nM) after 4 h (p70S6k) or 24 h incubation. An actin loading control for (C), (D) and (E) is shown in (F). A densitometric analysis representing the average from three blots is shown below each Western blot. Differences from control are shown as cP<0.001 compared with control; dP<0.05, eP<0.01 or fP<0.001 compared with pcDNA3(−).

The effect of Akt on total protein degradation was mirrored by the effect on the ‘chymotrypsin-like’ enzyme activity, the predominant proteolytic activity of the proteasome. Thus proteolytic activity in the presence of PIF (Figure 4B) was elevated in myotubes expressing MyrAkt over pcDNA3(−), whereas there was little or no increase in activity in myotubes expressing DNAkt. In addition, expression of both 20S proteasome α- and β-subunits (Figures 4C and 4D), as well as p42, an ATPase subunit of the 19S regulator (Figure 4E), and p70S6k (Figure 4G) showed a greater enhancement in myotubes expressing MyrAkt over pcDNA3(−), whereas there was no increase in expression in myotubes expressing DNAkt. Levels of myosin were also more significantly reduced in myotubes expressing MyrAkt than in empty plasmid (Figure 4F), whereas there was no decrease in myosin in the presence of PIF when the myotubes expressed DNAkt. As previously reported [33] for myotubes exposed to TNFα and IFN-γ, PIF reduced myosin expression by up to 60% in those expressing both MyrAkt and empty plasmid, whereas there was no appreciable loss in the expression of actin. These results suggest that activation of Akt appears to be essential for the induction of proteasome activity in the presence of catabolic stimuli.

Myotubes expressing MyrAkt showed a small, non-significant increase in protein synthesis over pcDNA3(−) over a 4 h period (Figure 5), whereas myotubes expressing DNAkt showed a significant reduction in protein synthesis. Myotubes exposed to PIF showed a reduction in protein synthesis with a maximal effect at 4.2 nM (Figure 5), as for the effect on protein degradation (Figure 4A), as previously reported [31]. However, there was no difference in the relative inhibition of protein synthesis in myotubes containing empty plasmid or in those expressing MyrAkt or DNAkt. This suggests that Akt is unable to reverse the depression in protein synthesis induced by PIF, which has been shown to be due to an increased phosphorylation of eIF2α [31].

Effect of PIF on protein synthesis after 4 h incubation in myotubes containing pcDNA3(−) (solid bars), Myr Akt (stippled bars) and DNAkt (open bars)

Figure 5
Effect of PIF on protein synthesis after 4 h incubation in myotubes containing pcDNA3(−) (solid bars), Myr Akt (stippled bars) and DNAkt (open bars)

Differences from controls are indicated as aP<0.05 or cP<0.001 compared with control; dP<0.05 or eP<0.01 compared with pcDNA3(−).

Figure 5
Effect of PIF on protein synthesis after 4 h incubation in myotubes containing pcDNA3(−) (solid bars), Myr Akt (stippled bars) and DNAkt (open bars)

Differences from controls are indicated as aP<0.05 or cP<0.001 compared with control; dP<0.05 or eP<0.01 compared with pcDNA3(−).

The effect of PIF both on protein degradation (Figure 6A) and the ‘chymotrypsin-like’ enzyme activity (Figure 6B) was attenuated by LY294002 in myotubes containing empty plasmid pcDNA3(−). However, LY294002 had no effect on either protein degradation or ‘chymotrypsin-like’ enzyme activity in myotubes containing constitutively active Akt (MyrAkt) (Figure 6). These results suggest that protein degradation, through an increase in proteasome activity in the presence of PIF, requires the PI3K-induced activation of Akt.

Effect of the PI3K inhibitor LY294002 on protein degradation, proteasome activity and degradation of IκBα by PIF

Figure 6
Effect of the PI3K inhibitor LY294002 on protein degradation, proteasome activity and degradation of IκBα by PIF

Effect of PIF on protein degradation (A) and ‘chymotrypsin-like’ enzyme activity (B) in myotubes containing pcDNA3(−) (◆) and MyrAkt (▲) in the absence (closed symbols) or presence (open symbols) of LY294002 (25 μM). Myotubes were preincubated with LY294002 2 h prior to the addition of PIF. Protein degradation and ‘chymotrypsin-like’ enzyme activity were determined 24 h after addition of PIF. Western blot analysis of the effect of PIF on phospho-IκBα (C) and IκBα (D) and total cellular actin (E) in myotubes determined 30 min after the addition of PIF in the absence or presence of 25 μM LY294002. The blots shown are representative of the three separate experiments. The densitometric analysis is shown below each blot for myotubes in the absence (solid bars) or presence [open bars in (C) and stippled bars in (D)] of LY294002. aP<0.05, bP<0.01 or cP<0.001 compared with control; dP<0.05, eP<0.01 or fP<0.001 compared with the presence of LY294002.

Figure 6
Effect of the PI3K inhibitor LY294002 on protein degradation, proteasome activity and degradation of IκBα by PIF

Effect of PIF on protein degradation (A) and ‘chymotrypsin-like’ enzyme activity (B) in myotubes containing pcDNA3(−) (◆) and MyrAkt (▲) in the absence (closed symbols) or presence (open symbols) of LY294002 (25 μM). Myotubes were preincubated with LY294002 2 h prior to the addition of PIF. Protein degradation and ‘chymotrypsin-like’ enzyme activity were determined 24 h after addition of PIF. Western blot analysis of the effect of PIF on phospho-IκBα (C) and IκBα (D) and total cellular actin (E) in myotubes determined 30 min after the addition of PIF in the absence or presence of 25 μM LY294002. The blots shown are representative of the three separate experiments. The densitometric analysis is shown below each blot for myotubes in the absence (solid bars) or presence [open bars in (C) and stippled bars in (D)] of LY294002. aP<0.05, bP<0.01 or cP<0.001 compared with control; dP<0.05, eP<0.01 or fP<0.001 compared with the presence of LY294002.

To investigate the possibility that activated Akt was also re-sponsible for the phosphorylation and the consequent degradation of IκBα, the effect of LY294002 on this process was determined. PIF induced an increase in IκBα phosphorylation (Figure 6C) and a reciprocal decrease in the cytosolic level of IκBα in myotubes (Figure 6D), and this was completely attenuated by LY294002. In addition, although myotubes containing empty plasmid (Figure 7A) showed the same response to PIF and LY294002 as untransfected myotubes (Figure 6C) there was no phosphorylation of IκBα and no degradation of IκBα in response to PIF in myotubes containing DNAkt, and no effect of LY294002 (Figure 7A). In contrast, myotubes containing MyrAkt showed degradation of IκBα in response to PIF, but this was not attenuated by LY294002. These results confirm that activation of Akt by PIF results in the phosphorylation and subsequent degradation of IκBα. Degradation of IκBα should result in an increased nuclear accumulation of NF-κB and the EMSA is shown in Figure 7(B). To confirm the role of Akt and the specificity of LY294002, nuclear binding of NF-κB was measured in transfected myotubes (Figure 7B). Thus PIF induced nuclear migration of NF-κB in myotubes containing empty plasmid [pcDNA3(−)], but not in those containing DNAkt. In addition, in myotubes containing MyrAkt, there was constitutive up-regulation of NF-κB, and LY294002 had no effect on nuclear binding of NF-κB in the presence of PIF. These results confirm that Akt is important in the activation of NF-κB through a PI3K-mediated process.

Role of Akt in phosphorylation and degradation of IκBα and nuclear translocation of NF-κB in the presence of PIF

Figure 7
Role of Akt in phosphorylation and degradation of IκBα and nuclear translocation of NF-κB in the presence of PIF

Effect of PIF on phospho-IκBα and IκBα (A), determined by Western blotting, and nuclear translocation of NF-κB (B), determined by EMSA, in murine myotubes containing empty pcDNA3 plasmid (wild-type), MyrAkt and DNAkt. Measurements were made 30 min after treatment with PIF (4.2 nM) in the absence or presence of 25 μM LY294002, added 2 h prior to PIF. The blots shown are representative of three separate experiments and the densitometric analysis is an average of the three blots. In (A) the densitometric analysis for wild-type (solid bars), MyrAkt (diagonally hatched bars) and DNAkt (horizontally hatched bars) is shown for both phospho-IκBα (pIκBα) and IκBα. aP<0.05 or cP<0.001 compared with control; eP<0.01 or fP <0.001 compared with in the presence of LY294002. hP<0.01 or iP<0.001 compared with the corresponding pcDNA3; jP<0.05 or kP<0.01 compared with the corresponding MyrAkt.

Figure 7
Role of Akt in phosphorylation and degradation of IκBα and nuclear translocation of NF-κB in the presence of PIF

Effect of PIF on phospho-IκBα and IκBα (A), determined by Western blotting, and nuclear translocation of NF-κB (B), determined by EMSA, in murine myotubes containing empty pcDNA3 plasmid (wild-type), MyrAkt and DNAkt. Measurements were made 30 min after treatment with PIF (4.2 nM) in the absence or presence of 25 μM LY294002, added 2 h prior to PIF. The blots shown are representative of three separate experiments and the densitometric analysis is an average of the three blots. In (A) the densitometric analysis for wild-type (solid bars), MyrAkt (diagonally hatched bars) and DNAkt (horizontally hatched bars) is shown for both phospho-IκBα (pIκBα) and IκBα. aP<0.05 or cP<0.001 compared with control; eP<0.01 or fP <0.001 compared with in the presence of LY294002. hP<0.01 or iP<0.001 compared with the corresponding pcDNA3; jP<0.05 or kP<0.01 compared with the corresponding MyrAkt.

DISCUSSION

The results of the present study provide evidence for a role of Akt in protein degradation in skeletal muscle in response to PIF and Ang II through an increased expression and activity of proteasome subunits. The mechanism of this effect appears to be through increased phosphorylation and subsequent degradation of IκBα, possibly through activation of IKK, leading to increased nuclear accumulation of NF-κB, which has been shown to act as a transcription factor for the induction of proteasome expression by PIF [7]. Thus transfection of myotubes with plasmids that had mutations at the serine phosphorylation site required for degradation of IκBα, which prevented nuclear accumulation of NF-κB, attenuated protein degradation induced by PIF, as well as induction of ‘chymotrypsin-like’ enzyme activity and proteasome subunits [34]. In addition, inhibitors of NF-κB activation, such as resveratrol, which inhibits IKK activation were shown not only to attenuate PIF-induced protein degradation in murine myotubes, but also protein degradation in skeletal muscle of mice bearing a cachexia-inducing tumour [35]. Also, activation of Akt appears to be necessary for the transitory stimulation of mTOR, leading to activation (phosphorylation) of p70S6k, which would confer selective translation of mRNAs that contain a 5′-polypyrimidine tract. Activation of Akt appears to occur through the PI3K pathway, which is known to be activated by receptor and non-receptor tyrosine kinases [36]. This may be a potential mechanism by which PIF could activate PI3K, since the tyrosine kinase inhibitors genistein and tryphostin 23 have been shown to attenuate PIF-induced proteasome expression [37]. Also genistein has been shown in the present study to attenuate the induction of Akt phosphorylation by PIF. This is the first study in which Akt has been shown to be involved in protein degradation in muscle. Akt has previously been shown to be involved in muscle hypertrophy, and inhibition of Akt, either by inhibition of PI3K, or expression of DNAkt reduces the mean size of myotubes in culture [38]. In the present study, in the absence of agonists, expression of DNAkt significantly reduced protein synthesis compared with wild-type myotubes, and expression of MyrAkt also increased protein synthesis, although this did not reach significance. However constitutive expression of Akt did not protect against the decrease in protein synthesis in the presence of PIF, which we have recently shown [31] to be due to an increased phosphorylation of eIF2α.

The results of the present study are in contrast with those of other workers, who have shown that IGF-I attenuates proteolysis in cultured L6 myotubes [39] or C2C12 myotubes [40] in the presence of dexamethasone, through a PI3K/Akt/mTOR-dependent mechanism. This may reflect the differing mechanisms by which glucocorticoids induce muscle atrophy which involves Akt-Foxo1 [41], although PIF and Ang II produce the same effect through activation of the transcription factor NF-κB [9,34,35]. However, the PI3K/Akt pathway may play a dual role in muscle, since it can also activate NF-κB. This has been shown to occur in skeletal muscle of both normal and dystrophin-deficient mice in response to mechanical stretch [42]. In the process, Akt activates NF-κB signalling by direct activation of the IKK complex, which then phosphorylates IκB releasing NF-κB to move into the nucleus. This is not the first study to show a role for PI3K in protein degradation. Fang et al. [43] showed that the PI3K inhibitors LY294002 and wortmannin reduced the rate of protein breakdown in muscles from burned rats, and Dardevet et al. [44] also showed that wortmannin reduced basal protein breakdown rates in incubated rat muscles. This suggests that the PI3K/Akt pathway may play a dual role in muscle, stimulating both hypertrophy and atrophy depending on the agonist.

Activation of Akt appears to be necessary not only for the activation of the transcription factor NF-κB, which might be expected to lead to increased mRNA levels for proteasome subunits and MuRF1 [11], but also for the subsequent protein synthesis, possibly through the mTOR/p70S6k pathway. We have shown [45] that protein synthesis is required for protein degradation, and the increased expression of proteasome subunits in the presence of PIF, which was blocked in the presence of cycloheximide. A number of other reports [1114] have shown an increased expression of mRNA for MuRF1 and atrogin-1/MAFbx in atrophying muscle, but there have been no studies as to how this is translated into protein in the environment of depressed protein synthesis. Both PIF and Ang II inhibit translation initiation through an increased phosphorylation of eIF2α, which would inhibit initiator methionine tRNA binding to the 40S ribosomal subunit [31]. In the present study rapamycin, a specific inhibitor of mTOR, attenuated the increased protein degradation induced by PIF when added before or up to 30 min after the agonist, suggesting a need for the mTOR pathway in early steps of this process. Activation of mTOR has been confirmed by Western blotting, which shows maximal phosphorylation within 0.5 h of PIF addition, and only significant elevation up to 1 h. This follows the same time course as PIF-induced phosphorylation of Akt. Activation of mTOR is followed by activation (phosphorylation) of p70S6k, which is significantly increased only between 1 and 2 h, suggesting a need for protein synthesis at this point leading to increased protein expression of proteasome α-subunits and E214k which is seen within 3 h of PIF incubation [45]. Acharyya et al. [46] have shown an increase in phosphorylation of p70S6k in gastrocnemius muscle of cachectic mice bearing the colon 26 tumour, although this was not accompanied by an increased phosphorylation of Akt. Thus Akt appears to play a dual role in the activation of the ubiquitin–proteasome pathway, whereas activation of PI3K is an important component of a signalling mechanism leading to transient ROS formation [15].

The results of the present study confirm the importance of NF-κB activation in the stimulation of protein degradation in murine myotubes and provide evidence for an alternative role for Akt in this process. Transitory activation of Akt appears to play an important role in degradation of myofibrillar proteins through the ubiquitin–proteasome pathway.

This work has been supported by Novartis Medical Nutrition (H.L.E.) and Ark Therapeutics Ltd (S.T.R.).

Abbreviations

     
  • Akt/PKB

    protein kinase B

  •  
  • AMC

    aminomethyl coumarin

  •  
  • Ang II

    angiotensin II

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • DNAkt

    dominant-negative Akt construct

  •  
  • ECL

    enhanced chemiluminescence

  •  
  • eIF2α

    eukaryotic initiation factor 2α

  •  
  • EMSA

    electrophoretic mobility-shift assay

  •  
  • FCS

    foetal calf serum

  •  
  • Foxo

    Forkhead box O

  •  
  • GSK

    glycogen synthase kinase

  •  
  • HA

    haemagglutinin

  •  
  • HS

    horse serum

  •  
  • IFN-γ

    interferon-γ

  •  
  • IGF-I

    insulin-like growth factor I

  •  
  • IκB

    inhibitory κB

  •  
  • IKK

    IκB kinase

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • MyrAkt

    constitutively active Akt construct

  •  
  • NF-κB

    nuclear factor κB

  •  
  • p70S6k

    70 kDa ribosomal protein S6 kinase

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PIF

    proteolysis-inducing factor

  •  
  • ROS

    reactive oxygen species

  •  
  • TNFα

    tumour necrosis factor α

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